Implants with attached silylated therapeutic agents

ABSTRACT

The present invention is directed to implants that include therapeutic molecules bonded to their surfaces. The therapeutic molecules interact with cells that are adjacent, near, or adhering to the implant. The covalently-bonded therapeutic molecules may be released from the implant surface by changes in pH or enzymes characteristic of cells adjacent to the implant. Preferably the covalently-bonded agents include an antibiotics that are released from the implant surface by bacteria and in this way ensures that the antibiotic is released at sites on the implant that would serve as centers for both bacterial colonization and biofilm formation.

BACKGROUND AND SUMMARY OF THE INVENTION

Implant-associated infections are an ever-present and devastatingcomplication of insertion of a foreign object, such as a stent,catheter, intravenous delivery tube (Hickman), heart valve, dentalimplant, electro-mechanical device, prosthetic device, glucose sensor,or stabilizing device such as orthopedic nails and pins. Sources ofthese infections include introduction of foreign bodies during wounding,introduction of microorganisms in the surgical suite, constant access toindwelling catheters, and hematogenous infections that arise at a sitedistal from the implant. Generally, infections associated with implantscurrently require systemic pharmaceutical treatment of the patient.

Open fracture wounds from ballistic injury are also a serious healthproblem and require on-site external fixation followed by internalfixation to allow the fracture to heal. In such wounds, the probabilityof infection is very high. The extended infection control procedures andsubsequent protracted healing time results in prolonged disability,often incomplete healing, and hospital costs.

Generally, principles of open fracture management include emergent andadequate wound debridement, often on multiple occasions, immobilization(often by external fixation), antibiotic therapy, and secondary closureor coverage of the wound. The definitive fracture management byintramedullary nail insertion is delayed until adequate soft tissuerepair has occurred. Open fractures in general, and high-energy gunshotwounds, in particular require immediate and aggressive irrigation anddebridement for removal of microscopic and macroscopic contaminants suchas clothing and bullet wadding. Nevertheless, despite immediate surgicalintervention open wounds often become infected. In animal studies asmany as 60% of wounds can be infected within 12 hours of such trauma.Importantly, the early administration of antibiotics inhibits bacterialgrowth associated with delays in debridement, while reducing subsequenttissue necrosis.

Periprosthetic infections after orthopaedic surgery are extremely costlyand frequently lead to prolonged disability. Improvements in steriletechnique have lowered the incidence to below 1% in many high throughputcenters. However, cases of post-surgical infections still are numerous.Each case requires a minimum of aggressive antibiotic treatment andoften at least one additional surgery. Unfortunately, for the populationmost frequently affected by such infections, i.e. the elderly, thediabetic, and the immunocompromised, such surgeries place the patient atgreat risk.

In general, implant-associated infections arise from two causes. Thefirst of these, the so-called hematogenous infection, is due to aninfection that is present elsewhere in the body. Bacteria from thisinfection opportunistically populate the protected environment aroundthe partially encapsulated/partially integrated implant. The secondcause of infections is the introduction of microorganisms duringsurgery. To minimize introduction of infection, very stringentconditions have been developed for the surgical site, which include useof space suits, filtered air, and laminar flow areas. Additionally,there is constant lavage of the surgical site with antibiotic solutionsand post-surgical systemic prophylaxis.

Irrespective of the etiology, the patient can be aggressively treatedwith antibiotics and/or surgery. For a periprosthetic infection, thesurgery minimally involves debridement of the bone to remove adherentmicroorganisms and replacement of the polymer parts. More aggressiveprocedures are employed in many circumstances, including placement of aspacer block that can deliver therapeutic doses of antibiotic tostabilize the cavity or use of antibiotic-releasing cement to anchor theprosthesis. Commonly, these regimens result in a compromised bone stock;in recalcitrant infections, arthrodesis or amputation may be required.Thus, any approach to limit or even prevent implant-associatedinfections in general and orthopaedic infections in particular can havea large impact on reducing healthcare costs, minimizing pain andsuffering, and improving the quality of life of the affected patients.

Surgical implantation of a prosthesis results in formation of a fibrousclot, followed by fibrous encapsulation of the foreign object withvarying degrees of inflammation. Subsequently, as much as 90% of theimplant is surrounded by a fibrous membrane. Unfortunately, theencapsulation by the fibrous membrane provides a protectedmicroenvironment that is ideal for bacterial propagation. Initial stagesof bacterial colonization involve the switch from a mobile, planktonicbacterium to one that is adherent to the inert surface. As the numbersof bacteria become greater, they organize into colonies that can formlarge biofilms. These sites on the implant surface are poorly accessedby the immune system and are chemically protected from antibiotics.

The majority of periprosthetic infections are caused by Staphylococcusaureus, and S. epidermidis, both gram-positive bacteria; less frequentinfections are caused by the gram-negative organisms. Both S. aureus andS. epidermidis are commonly present in the operating room environmentand are implicated in infections involving prostheses, stents and otherbiomaterial implants. Both species adhere to the biomaterial surfaces,propagate rapidly, and during this proliferation, generate a pre-biofilmslime. Production of the polysaccharide-enclosed clumps of bacteria,characteristic of a biofilm, completes the process of restrictingantibiotic access to the bacterial surface. This biofilm can effectivelyimmobilize many antibiotics thereby reducing the numbers of therapeuticmolecules that can penetrate and interact with the bacteria. Both S.aureus and S. epidermidis form such biofilms. When biofilms are formed,surgical experience dictates complete removal of the prostheticcomponents and debridement. Clearly, if antibiotics can be released atan early colonization or pre-colonization stage, the biofilm formationcan be impeded and the infection can be extinguished, obviating the needfor surgical intervention.

Infection following joint arthroplasty is a devastating complicationwith immense financial and psychological costs. Effective measures,including the use of body exhaust systems, laminar airflow, prophylacticantibiotics, and various other precautions, have been successful inreducing the incidence of periprosthetic infection. Despite all thesemeasures, deep infection still occurs after 1-5 percent of jointreplacements. The incidence is even higher in some ‘at risk’ patientssuch as diabetics, patients with remote history of infection, and thosewith inflammatory arthropathies. Generally, principles of periprostheticinfection management include emergent debridement and irrigation inselect cases, resection arthroplasty and delayed reimplantation untileradication of the organisms has occurred. Prolonged antibiotictreatment in concert with multiple surgeries results in extended periodsof patient disability. A multitude of factors influence the success ofthese surgical interventions. The most important relate to the efficacyby which antibiotics are delivered to the local tissues and theosteogenic potential of the surrounding bone.

Infection prevention/treatment in patients with implants requires robustregiments that may include systemic antibiotic treatment and use ofimplants that achieve a controlled antibiotic release, usually in theimmediate post-operative period. It would be desirable to provide for animplant that maintains a reservoir of antibiotics or other therapeuticmolecules that can be activated at the time of an establishinginfection, whether in the immediate post-operative period or at longertimes after implantation.

SUMMARY

The present invention is an implant that includes therapeutic moleculeslike antibiotics bonded to the implant surface and that interact withcells that are adjacent, near, or adhering to the implant. Thetherapeutic molecules may be bonded to the implant surface by anacid-labile linkage; a covalent linkage that includes a cellmembrane-soluble portion between the implant and the tetheredtherapeutic molecule; a covalent linkage connected to a ligand that isthen loaded with the therapeutic molecule through its high affinitybinding to its ligand, and/or an enzyme-labile linkage. Enzymes, acids,or endogenous ligands characterizing the cells or their activities mayinteract with or cleave the bonded therapeutic molecules on the implant.The therapeutic molecules may be used to alter the proliferation ofthese cells and the implant may be used to promote the adhesion andmaturation of these cells. The surface modified implant provides areservoir of therapeutic molecules that are controllably released inresponse to the cellular environment of the implant.

One embodiment of the present invention is an implant for a mammal. Theimplant has a biologically compatible surface with at least a portion ofthe surface silylated with organosilanes. One or more of theseorganosilane may be bonded to therapeutic molecules with the therapeuticmolecule interacting with cells adjacent to the surface of the implant.The bond between the organosilane and the therapeutic molecules may be acovalent bond, and can include an acid-labile or enzyme-labile bond suchthat the therapeutic molecule is released from the organosilane byinteracting with enzymes or acids produced by the cells. The therapeuticmolecule may also be competitively bonded to the organosilane andreleased by exchange with endogenous ligands of the cell. Thetherapeutic molecules may include peptides, therapeuticoligonucleotides, antibiotics, cell growth factors, chemotherapeutics,thrombolytic, anti-inflammatories, osteoactive factors and combinationsof these molecules. The therapeutic molecules covalently bonded to theimplant may be used to alter angiogenesis, or decrease bacterialproliferation in cells or tissue adjacent to the implant. Preferably thebioactive surface remains active until the danger of disease has passed.

One embodiment of the present invention is an implant for a mammalhaving a biologically compatible surface. At least a portion of thesurface is silylated with organosilanes and one or more of theorganosilanes is covalently bonded to a first end of a linking group.The second end of the linking group may be bonded to therapeuticmolecules that interact with cells adjacent to the surface of theimplant. The therapeutic molecule may include peptides, therapeuticoligonucleotides, antibiotics, cell growth factors, chemotherapeutics,anti-thrombolytic, anti-inflammatories, osteoactive factors, andcombinations of these. The therapeutic molecule may be bonded to alinking group that can enter or pass through the membrane or the wall ofcells. The linker bonded to the organosilane can include amino acids, apeptide, or spacers like oligo(ethylene glycol). The linker may alsoinclude an acid labile moiety methylmaleamide, hydrazone, andcombinations of these. Labilization of covalently bonded therapeuticmolecules from the linker by cellular acid or enzymes releases thetherapeutic molecule from the implant to interact with the cells andpreferably leaves a peptide covalently bonded to the surface that may beused to promote the adhesion and maturation of cells on the implant. Thetherapeutic molecule can be competitively bonded to the linker and maybe exchanged with endogenous ligands of the cell. The therapeuticmolecules bonded to the implant may interact with the cells to alterangiogenesis, or decrease bacterial proliferation adjacent to theimplant.

One embodiment of the present invention is an implant for use in amammal that has a first terminal end of one or more linking groupscovalently bonded to a silylated implant surface. The second, oropposite, terminal end of the one or more of the linking groups can bebonded to an antibiotic that is used to interact with cells adjacent tothe surface of the implant. The linking group bonded to the implantsurface preferably includes an integrin binding peptide sequence and caninclude an acid or enzyme-labile bond such that labilization of theantibiotic molecule from the linker provides a peptide that promotes theadhesion and maturation of bone cells. The antibiotic may be tethered tothe implant through a long, flexible partially membrane-soluble linkeror the antibiotic may be competitively bonded to the linker and releasedby competitive binding with endogenous ligands of the cell. Theantibiotic may be active against gram negative bacteria, active againstboth gram-positive and gram-negative bacteria, and preferably theantibiotic is active against gram positive bacteria. The antibiotic mayinclude but is not limited to minocyclins, tigecyclin, glycylcycline,vancomycin and its analogs, rifampin and its analogs, gentamycin and itsanalogs, or combinations thereof.

One embodiment of the present invention is a method of treating a mammalthat includes inserting an implant into a site such as a fracture, anartery, or body cavity of the mammal. The implant has a biologicallycompatible surface that is silylated with organosilanes and where one ormore of the organosilanes is bonded to therapeutic molecules. Thetherapeutic molecule interacts with cells adjacent to the surface of theimplant in the mammal. The therapeutic molecule may remain bonded to theimplant and interact with the cells or they can be released from theorganosilane by reaction with a cellularly derived acid, enzyme, orligand. The therapeutic molecules from the implant can alter theproliferation of cells at the site of the implant. Preferably theimplant promotes osseointegration during fracture fixation and thetherapeutic molecules bonded to the implant include antibiotics toprevent bacterial proliferation at a fracture fixation site. Thesurface-modified implant, by virtue of the tethered antibiotics, can beused to inhibit bacterial proliferation after the initial adhesion ofthe bacterium to the implant surface in a mammalian patient.

In a preferred embodiment antibiotics can be covalently bonded orcomplexed to a metal such as titanium by organosilane linkers that havesites or moieties that are degraded enzymatically or are labile undervarious pH conditions. In a preferred embodiment, agents released frombacteria near or adhering to an implant can cleave an antibiotic-linkerbond so as to release high focal concentrations of an antibiotic fromthe implant, killing susceptible bacteria. Even more preferably, thesurface modifications on the implant lower bacterial counts whilesustaining osteoblastic cell proliferation.

The overall activity of these modified implant surfaces can be tailoredso that they react at the earliest stages of infection as the bacteriabegin to colonize the implant surface. Preferably the adherent bacteriaencounters a surface that can either release antibiotic from the linkerdue to local pH changes associated with the proliferating bacteria,release antibiotic by linker cleavage due to cellular enzyme release, orrelease antibiotic by competition from a linkage by an endogenous ligandof the cell. Alternatively, the cells may internalize the antibioticbecause of the membrane-solubility of a linker. This internalizedantibiotic may be active while still bound to the implant, as is thecase for vancomycin. Alternatively, where the enzyme to cleave thebioactive molecule resides in the bacterial wall, in the cell membrane,or in the cellular milieu, an enzyme associated with the bacteria orwith the cell cleaves the bioactive moiety from the linker to release itwithin the cell. This directed release of antibiotic would result inpreferential killing of adherent bacteria, i.e. those that arecolonizing the surface to initiate biofilm formation.

The release of therapeutic molecules such as antibiotics from themodified implant surface occurs as a function of bacterial colonizationand thus the bioactive moieties remain latent until activated by thepresence of bacteria. Such a device can be used for the prevention ofimplant-related infections, both immediately after the implant isinserted as well as for the longer term prevention of hematogenousinfections.

By prevention of bacterial colonization of the implant surface, anyremaining bacteria are accessible for clearance by the immune system.The implant surface modifications of the present invention are inertunder conditions where their activity is not required and are activewhen bacteria begin to foul an implant. It has a clear advantage overthe existing technologies in that the surface does not release activeantibiotics until cued to do so by bacterial adhesion. Moreover, thesemodified implants can be formulated to be stable for long periods, thushaving a ready reservoir of antibiotics to reduce the risks of secondaryinfections. The surface-modified implants of the present invention mayalso be made to include other therapeutic molecules tethered to thesurface for the treatment of conditions such as cancer, restenosis, boneloss, and thromboses.

One use for the present invention is for the modification of implantsurfaces with antibiotics that are most frequently fouled by biofilmformation, such as prosthesis and nails used for fracture fixation andsurfaces of indwelling catheters. The bactericidal surface could also beused to modify implant surfaces in less high-risk implant situations,such as the orthopaedic arthroplasties and stent placement whereinfection is infrequent, but once established can have devastatingconsequences.

DESCRIPTION OF THE DRAWINGS

In part, other aspects, features, benefits and advantages of theembodiments of the present invention can be apparent with regard to thefollowing description, appended claims and accompanying drawings where:

FIG. 1 Illustrates the general chemical structure of an implant surfacemodified by covalent bonding of molecules to its surface;

FIG. 2 MALDI-TOF mass spectroscopy of putativeVAN-AEEA-AEEA-NH—Pr—Ti6Al4V from a 2,5-dihydroxy-benzoic acid (2,5-DHBA)matrix;

FIG. 3 Ti-tethered vancomycin kills adherent S. aureus. Vancomycin,tethered to the Ti surface, was incubated with S. aureus for 1 h andstained for bacterial viability. Live bacteria stain green (lightgray)-note the small number of live bacteria on the vancomycin tetheredsurface (C) (VAN-OEG-APTS-Ti). Dead bacteria stain red (light gray);note the larger number of dead bacteria on the vancomycin-tethered tothe Ti surface (D);

FIG. 4 A schematic diagram of the antibiotic17-(2-methyl-4-thioethylamido maleyl)rifampin modified at 17-OH forbonding with an implant surface silylated with MPTS to form a disulfideor GPTS to form a thioether;

FIG. 5 (A) is a schematic illustration of the bioactive moleculevancomycin, whose linkage to the metal implant is labile to acid,reacting with acid secreted by bacterium near the implant surface; (B)illustrates the released vancomycin from the modified implantinteracting with the bacterium;

FIG. 6 (A) is a schematic illustration of the bioactive moleculevancomycin irreversibly tethered by a covalent linkage to the implantsurface; (B) illustrates the passage of the bioactive molecule throughthe cell wall using the membrane soluble linkage that places vancomycinat its site of action in the cell;

FIG. 7 (A) is a schematic illustration of the bioactive moleculevancomycin bound to an affinity ligand connected to the implant surface;(B) illustrates the vancomycin is released by competitive binding withits native substrate in the bacterium;

FIG. 8 Illustrates the chemical structure of linkages bonded to theimplant surface represented by R₁ and the acid labile moieties maleamideand hydrazone; groups R₂ and R₃ represent therapeutic molecules;

FIG. 9 (A) is a schematic illustration of the antibiotic tigecyclinlinked to Ti-RGD (SEQ ID NO: 1) by acid-labile linkages that are cleavedby acid secreted by an adjacent bacterium; (B) illustrates releasedantibiotic interacting with the bacterium and that the releasedtigecyclin has exposed the RGD (SEQ ID NO: 1) motif bonded to theimplant surface;

FIG. 10 (A) is a schematic illustration of a modified implant surfacewith covalently bonded acid-labile tigecyclin bioactive therapeuticmolecules bonded to a portion of the RGD (SEQ ID NO: 1) peptides linkedto the implant surface; (B) is a schematic illustration of the modifiedsurface releasing tigecyclin in response to acid released by a cell; (C)is a schematic illustration of the modified implant surface illustratingosteoblast cell attachment, integrin clustering, and extracellularmatrix protein deposition.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention that can be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The present invention is directed to bonding therapeutic molecules tothe surface of implantable substrates that used for fracture fixation,prosthetics, catheters, or monitoring sensors in mammals. Thetherapeutic molecules may be bonded to the implant surface by linkagesthat release the molecules in response to cellular activity by the cellsnear the modified implant surface.

A schematic diagram of a non-limiting embodiment of the presentinvention where a therapeutic molecule is bonded to the surface of animplant is shown in FIG. 1. The implant surface is silylated using anorganosilane which has an organic group that may be used to couple othermolecules to the implant surface. The organic group may include but isnot limited to one or more amine groups, epoxides, carboxylic acids, orthiol groups. Silylation of the implant is represented by the Ti—O—Si(CH₂)₃NH portion (A) of the structure in FIG. 1. The first end orterminus of a linking group is covalently bonded to the organic group ofthe organosilane. As shown in FIG. 1, this may be coupling of the aminoend of the organosilane with the carboxyl end of a representative linkerwith a cell adhesion sequence that includes amino acids forming thepeptide sequence RGDS (SEQ ID NO:2), portion (B). Other linkers mayinclude different amino acids, the use different sized flexibleoligo-ethylene glycol spacers, or a different reactive group at theterminal ends of the linking group. The second or opposite terminus endof the representative linker, in FIG. 1 it is an amino end however othergroups may be used, is bonded to a representative labile linkage. InFIG. 1 the acid-labile methylmaleamide linkage moiety is shown asportion (C). Other labile linkages may be used including but not limitedto enzyme-labile linkages. A representative therapeutic molecule (D),tigecyclin in FIG. 1, is bonded to the acid-labile moiety portion (C).This therapeutic molecule interacts with cells adjacent to the surfaceof the implant. Although FIG. 1 illustrates various portions (B), (C),and (D) of the modified titanium implant surface, the present inventionis not limited to this structure. For example the segments (B), (C) and(D) may be covalently linked together as a single unit and coupled tothe silylated implant surface. Alternatively, the labile group may becoupled directly to the organosilane or the therapeutic molecule mightbe covalently bonded directly to an organosilane and this molecule usedto silylate the surface of the implant.

In embodiments of the present invention, therapeutic molecules arebonded to the surface of an implant. The bonds connecting the linker andorganosilane to the implant surface are preferably covalent bonds. Thetherapeutic molecules may be bonded to the linker or organosilanethrough covalent or competitive bonding. The bond between the implantsurface and the therapeutic molecule may include a labile bond from alinker to the therapeutic moiety. This bond may be cleaved by asecretion by a cell which would include acids and enzymes. Thetherapeutic molecule would be released in the immediate vicinity of thecell that released the secretion to target its activity to that cell asshown schematically in FIG. 5 where the antibiotic vancomycin istethered to a Ti surface through an acid-labile linker. A stablenon-labile bond from the linker to the therapeutic moiety, such as anoligo(ethyleneglycol) chain spacer, would allow the therapeutic moleculeto enter or pass through the cell wall or cell membrane to be presentedinto the cellular space. In this case the therapeutic moiety remainsactive in its tethered state, such as for example the antibioticvancomycin as shown schematically in FIG. 6. Alternatively, thetherapeutic molecule may be competitively bonded to linking groups ororganosilanes that are covalently bound to the surface of the implant.The therapeutic molecule competitively bonded to the linking groups ororganosilanes may then pass through a cell wall or membrane. Endogenousligands of the cell competitively bind to the linking groups ororganosilanes releasing the therapeutic molecule within the cell asillustrated schematically for vancomycin in FIG. 7. The surface modifiedimplant may also include a linker or organosilane with a labile bondthat is adjacent to the therapeutic molecule and that is capable ofspanning a cell wall or cell membrane. The labile bond of the linkerwould be cleaved by intracellular enzymes, thereby releasing the drug orpeptide within the cell allowing it to have its specific effect. Anon-limiting example of such an intracellular enzyme would be a kinaseinhibitor.

The therapeutic molecule may be tethered or bonded to the implantsurface through an organosilane or through a linking group bonded to theorganosilane. The linker or organosilane may have an acid-labile site,an enzyme cleavage site, recognition sequences for metalloproteinases,plasmin, thrombin, or tissue plasminogen activator. The linker ororganosilane can be a long flexible partially membrane soluble chainthat is covalently bonded directly to the therapeutic molecule. Forexample, organosilane groups may be coupled to oligo(ethylene glycol)linkers on the implant surface and the other end of the linker used tocovalently bond to a therapeutic molecule such as vancomycin as shown inFIG. 6. Alternatively the flexible partially membrane soluble chain mayrelease the therapeutic molecule within the cell through a high affinitycompetitively bonded tethered ligand such as but not limited tosequences such as (D-Ala-D-Ala) as shown in FIG. 7, or by incorporatingan enzyme cleavage site into the flexible chain. Cleavage of acid-labileor enzyme-labile linkers and organosilanes to release the therapeuticmolecules can expose a peptide sequence, such as RGD (SEQ ID NO: 1),which supports cellular proliferation.

Based on a therapeutic molecule such as an antibiotic occupying ˜40 nm²of space, if an adherent bacterium or other cell occupies 1 μm², itcould have ˜25,000 therapeutic molecules of antibiotic tethered forrelease directly at the bacterium's surface. The concentration oftherapeutic molecules tethered to the implant surface may be increasedby bonding the bioactive molecule to the interstices of a porous implantsurface or within the interconnected regions to allow both increasedeffects of local concentrations, increased therapeutic moleculecapacity, as well as increased surface area for the additionaltherapeutic molecules. Multi-functional linkages may also be used totether more than one therapeutic molecule per site. Such branchedlinkers can serve to tether multiple antibiotic or other therapeuticmolecules to the implant surface through a single bond between thesurface and the linker.

The linker or organosilane may be a flexible membrane soluble chain suchas but not limited to oligo-ethylene glycol (OEG), and may also includenaturally occurring and non-naturally occurring amino acids and peptidesequences including such amino acids. Oligo-ethylene glycol terminatedsurfaces exhibit resistance to protein absorption and may have from 2 to20 repeat units. As shown schematically in FIG. 10, a silane linkage maybe used to tether or bond adhesion-enhancing peptides such as RGD (SEQID NO: 1) to a Ti containing implant substrates that are linked totherapeutic molecules like tigecyclin. The one or more therapeuticmolecules modifying the surface of the implant may be bonded to all or aportion of the surface of the implant through the linkers or theorganosilane. One or more different linkages or organosilane may be usedon different portions of the implant surface and mixtures of linkagesand organosilanes may be used to modify the implant surface as well. Thelinkages and organosilane may include amino acid and peptide sequenceswhich interact with cells, for example but not limited to RGD (SEQ IDNO: 1), and RGDS (SEQ ID NO: 2), or they may include non-peptidelinkages such as AEEA, polyethylene glycol (PEG), and OEG. Additionalpeptide sequences useful as linkers in the present invention includethose used for ECM peptidomimics such as RGES (SEQ ID NO: 3), DGEA (SEQID NO: 4), and EGEA (SEQ ID NO: 5) as well as the bioactive peptidesdisclosed in U.S. Pat. No. 6,428,579, the contents of which areincorporated herein by reference in their entirety.

The therapeutic molecule can be but is not limited to peptides,therapeutic oligonucleotides, or molecules like antibiotics,anitfungals, thrombolytics, anti-inflammatories, osteoactive factors, orchemotherapeutics. The therapeutic molecule should remain attached tothe implant surface in the physiological range near 7.4 for the life ofthe implant, until the danger of disease has passed, or until it isreleased by cellularly-derived secretions or active cellular proteinssuch as a generated acid or an enzyme. Acid-labile moieties in thelinkages or organosilanes bonded to the therapeutic molecules mayinclude but are not limited to an amide linkage, an ester linkage,methylmaleamide, or a hydrazone linkage. The methylmaleamide andhydrazone linkages are stable at physiological pH, but are readilyhydrolyzed in mild acid, pH 5-6. Preferably the acid-labile linkagepermits release of about 50% of the therapeutic molecules in the first60 min at a pH of between 5 and 6. Preferably the enzyme cleavablelinkage permits release of about 50% of the therapeutic molecule in thefirst 60 minutes at a desired enzyme concentration. In a preferredembodiment the modified implant surface supports osteoblastproliferation and maturation in the presence and absence of low-levelbacterial infection. A linker containing oligo(ethylene glycol)terminated by the vancomycin ligand, D-Ala-D-Ala (Ti—Si-Ala) may be madeas illustrated in FIG. 7. Vancomycin has a high affinity for its ligandand in this way could be bound to the surface via formation of a complexwith the ligand. In the presence of bacteria, the oligo(ethylene glycol)linker would allow the ligand-antibiotic complex to traverse the cellwall and the vancomycin would be competitively released to inhibitpeptide-glycan synthesis. Other suitable antibiotic/affinity ligandcomplexes may also be tethered to the implant surface; combinations ofantibiotics and various linkages on the implant surface may also bemade.

Substrates of the present invention are biologically compatiblematerials and which form condensation products with organosilanes. Theseimplant materials may include but are not limited to prosthetic,surgical, dental, and orthopedic alloys that include titanium and itsalloys and is preferably Ti6Al4V. Implants may also have layers thatinclude titanium that has been sputtered or vapor deposited onto theimplant. Other useful materials include but are not limited to tantalum,CoCrMo alloys, stainless steels, cobalt, chromium, aluminum, zirconium,gold, silicon and their alloys, Teflon/PTFE, polyethylene, includingultra high molecular weight polyethylene, silicone, silica, glass, andpolystyrene. Ceramic materials such as zirconia may also be used asimplants. Preferably the biologically compatible implant materials havesurfaces that can form condensation products with organosilane. Thesematerials may be hydrated, have one or more hydroxide groups on theirsurfaces, or may be treated to form such surfaces. These materials maybe molded, machined or shaped for a particular implant application andmaybe monolithic, a porous materials such as foams, sintered beads,woven surfaces, meshes, and other materials designed to increase surfacearea, porosity, fluid exchange and interconnectivity.

Therapeutic molecules of the present invention may be covalently bondedto the surface of the implant using organosilanes to form modifiedsilylated surfaces on the implant. Organosilanes, R_(n)SiX_((4-n)),useful in the present invention include those where the hydrolyzablegroup X is halogen, alkoxy, acyloxy or amine and R is a nonhydrolyzableorganic radical that posses a therapeutic molecule or a chemicalfunctionality that may be use to later covalently bond to a therapeuticmolecule or a linker. The organosilane forms stable condensationproducts with materials having oxide and or hydroxide groups on theirsurfaces. While not wishing to be bound by theory, one or more covalentbonds from each silicon of the organosilane forms a bond with thesubstrate surface as schematically illustrated in FIG. 1. A monolayer ofthe organosilane on the surface is preferred; however, multiplayerpolysiloxanes may be formed depending upon the concentration of theorganosilane used. Preferred organosilanes include but are not limitedto those where R includes an amine, a carboxylic acid, epoxide, cyanide,or sulfhydryl group. Examples of such organosilanes include but are notlimited to 3-aminopropyltriethoxysilane (APTS),3-carboxypropyltriethoxysilane (CPTS),(3-glycidoxypropyl)trimethoxysilane (GPTS), and3-mercaptopropyltrimethoxy silane (MPTS). Carboxylic acid terminatedimplant surfaces may also be formed by treatment of APTS surfaces withsuccinic anhydride. For example, APTS silylated implant substrates canbe prepared by etching the substrates with phosphate fluoride and thentreated with 0.2-1.0 mM 3-aminopropyltriethoxysilane (APTS) in hexanefor 45-90 min to silylate the surface The implant substrates can then besonicated in a hexane bath to remove excess reactants. The amino orcarboxy substrates can provide the base for Fmoc coupling of linkers,amino acids and therapeutic molecules such as vancomycin.

The activity of cells, their secretions, or other physiological eventsin a mammalian patient may be used to trigger the release of therapeuticmolecules, peptide, or drugs from the surface modified implants of thepresent invention. Non-limiting examples of such secretions or cellularevents include the acid released by bacteria, metalloproteinasescharacteristic of cancer cells, enzymes from clots associated with bonynon-unions, enzymes from clots associated with stents, and drug releasepredicated upon a specific event such as altered insulin level, glucoselevel or prostaglandin level. To be more specific, in the case of cancercells, one novel application is to the bony tumors arising frommetastases or from osteosarcomas. In this case, an implant would bemodified with tethered chemotherapeutic agents that can be released uponsecretion of high concentrations of metalloproteinases, enzymes that arecharacteristic of metastatic tumors and used for extracellular matrixdegradation. This device could then serve as an active defense againstre-establishment of the tumor at that site. In the case of bonynon-unions, again, an implant with tethered bone-inducing factors, suchas the bone morphogenetic proteins (BMP's) can be engineered to releaseBMP's upon dissolution of the blood clot associated with theimplantation, again using enzyme activities associated with thedegradation of the clot, such as the plasmin sequence. Conversely,implants could be engineered to release the growth factor during theinitial stages of osteolysis to prevent implant loosening. Additionally,clot formation within a stent could be inhibited through release ofthrombolytic drugs such as tissue plasminogen activator or anistreplaseat the site of clot formation.

To minimize development of patient resistance to antibiotics during acourse of treatment, many antibiotics such as rifampin are commonlygiven as a combination therapy with other drugs, such as isoniasid orethambutol. Additional drugs can be attached by covalent bonding to atether also chemically bonded to the implant with the antibiotic orchemotherapeutic to allow maximal efficacy in the treatment ofperiprosthetic infections or cancers.

Several factors have been identified in the mechanism ofosseointegration and bone ingrowth that lead to long-term implantstability. Osteoblast-like cells adhere more rapidly to metallicsurfaces than to standard polystyrene tissue culture surfaces and therate of adhesion is related to the roughness of the surface (porouscoating>rough>grit polished). Enhanced cell adhesion to the implant isaccompanied by increased osteoblastic maturation, matrix production andmineralization. Surface chemistry plays a role in osteoblastic celladhesion (Ti6Al4V>CoCr). Cell adhesion enhancement appears to bemediated by increased expression of the matrix protein, fibronectin, andits cognate membrane receptor, α5β1 integrin. The fibronectin-integrininteraction likely serves a subcellular signaling role as well asmediating adhesion, and osteoactive factors, TGF-β1 and BMP-2, stimulateosteoblast adhesion via enhanced fibronectin and α5β1 integrinexpression. Preferably the implant surfaces of the present inventionincorporate such materials, morphology, and factors onto the surfaceswhere possible.

Vancomycin is a therapeutic molecule that may be tethered to animplantable substrate surface, for example a Ti surface, using aflexible linkage that is not cleaved and places the vancomycin withinthe cell (Ti—O—Si—OEG-VAN) as shown in FIG. 6. This linkage can containvancomycin tethered to an oligo(ethylene glycol) linker that thencontinues into a flexible linker for attachment to the Ti surface.Vancomycin inhibits bacterial cell wall synthesis by inhibitingpeptidoglycan synthesis, a process that occurs on the exterior surfaceof the cell membrane. Membrane-anchored vancomycin would be expected todisplay an increased activity against resistant organisms. Immobilizedvancomycin on a flexible, non-labile hydrophobic linker, could bebactericidal without release from the Ti surface. The presence of theoligo(ethylene glycol) linker adjacent to the vancomycin can allowpassage through the bacterial cell wall while still maintaining contactwith the Ti surface as shown schematically in FIG. 6.

Tigecyclin is a therapeutic molecule that is an antibiotic that may betethered to a Ti implant surface. Tigecyclin is a glycylcycline derivedfrom minocycline with potency against tetracycline-sensitive and-resistant bacteria and thus is active against a broad spectrum ofgram-positive and gram-negative bacteria. It acts by inhibition of the30S ribosomal subunit, thereby disrupting protein synthesis. In vitro,tigecyclin has an [MIC90] of less than 0.25 mg/mL for most strainsincluding S. aureus; for E. faecalis and E. faecium, this low MIC iscoupled with an activity that was ˜8-fold more active than linezolid and32-fold more than quinupristin-dalfopristin. Using a pH-sensitivelinkage, tigecyclin may be tethered to a linker like an RGDS (SEQ ID NO:2) or RGD (SEQ ID NO: 1) peptide and the acid labile moietymethylmaleamide on an implant such as a Ti alloy surface (Ti—O—Si—RGD(SEQ ID NO: 1)-mTIG) as shown in FIG. 1. This methylmaleamide can becleaved under mildly acidic conditions (pH 5-6) and tigecyclin isreleased into the bacterial slime where it can kill the adheredmicroorganisms. Cleavage of the acid-labile linkers to release theantibiotic will expose the RGD (SEQ ID NO: 1) sequence as illustrated inFIG. 9(A) and FIG. 9(B), and support osteoblast proliferation.Extracellular acid generated by bacteria can cleave the acid-labilemaleamide bond, resulting in the release of tigecyclin at the bacterialcell wall. The infection is eliminated at its source in a time- andsite-specific manner. The antibiotic may be active against gram-negativebacteria, active against both gram-positive and gram-negative bacteria,and preferably the antibiotic is active against gram-positive bacteria.Other therapeutic antibiotic molecules bonded to the implant surface mayinclude but are not limited to minocyclins, Tigecyclin/GAR-396, thetetracyclin and glycylcycline antibiotics, vancomycin and its analogs,Rifampicin and its family members, Methcillin and its analogs,Gentamycin and its analogs and combinations of these.

The therapeutic molecule rapamycin is a macrocyclic triene antibioticproduced by Streptomyces hygroscopicus that binds to the cyclophilinreceptor and has been shown to inhibit the proliferation of vascularsmooth muscle cells. Rapamycin may be utilized in treating intimalsmooth muscle cell hyperplasia, restenosis, and vascular occlusion in amammal, particularly following either biologically or mechanicallymediated vascular injury. Such injury initiates a thrombotic andinflammatory response. Cell derived growth factors such as plateletderived growth factor, basic fibroblast growth factor, epidermal growthfactor, thrombin, etc., released from platelets, invading macrophagesand/or leukocytes, or directly from the smooth muscle cells provoke aproliferative and migratory response in medial smooth muscle cells.Daughter cells migrate to the intimal layer of arterial smooth muscleand continue to proliferate and secrete significant amounts ofextracellular matrix proteins. Rapamycin functions to inhibit smoothmuscle cell proliferation and does not interfere with there-endothelialization of the vessel walls. These secretions could beused to trigger release of rapamycin bonded to an implant surface ofsuch as a stent to prevent or restinosis.

In addition to being an effective anti-coagulant, heparin has also beendemonstrated to inhibit smooth muscle cell growth in vivo. Thus, heparinmay be effectively utilized in conjunction with rapamycin as atherapeutic molecule bonded to an implant surface in the treatment ofvascular disease. Essentially, the combination of rapamycin and heparinmay inhibit smooth muscle cell growth via two different mechanisms inaddition to the heparin acting as an anti-coagulant.

Antiinflammatory molecules may also be bonded to the implant substrateby reaction with silanized implant surfaces. Preferably thenon-steroidal anti-inflammatory molecules include those which inhibitthe synthesis of prostaglandins. Antiinflammatory molecules include butare not limited to acetylsalicyclic acid, indomethacin, ibuprofen,acetaminophen, apazone, celecoxib, and rofecoxib.

Oligonucleotides may also be bonded to the surface of an implant eitheralone or in combination with other therapeutic molecules. For example,RGD (SEQ ID NO: 1) peptide motifs may serve as part of the linker forthe immobilization of therapeutic oligonucleotides to the implant. TheRGD (SEQ ID NO: 1)-oligonucleotide linkage may be designed to be labileto matrix metalloproteinases released by cancer cells, thereby exposingadditional RGDs (SEQ ID NO: 1) for promotion of osseointegration as thetherapeutic oligonucleotide is released to the cancer cells adjacent tothe implant. Peptide sequences specifically cleaved by matrixmetalloproteinases can be placed between the RGD (SEQ ID NO: 1) motifand the therapeutic oligonucleotide. The effect of various amino acidsin linking peptides on the release of the therapeutic oligonucleotidesbonded to the implant may be determined by progressively changing thenumber and type of flanking amino acids at each side of the RGD (SEQ IDNO: 1) motif and anchor on the release of oligonucleotides covalentlybonded to the Ti surface.

Therapeutic oligonucleotides that may be bonded to the silylated implantsurface may include but are not limited to those anticanceroligonucleotides targeted against the MYC oncogene. Such therapeuticoligonucleotides may be composed of various combinations of modifiedbases, modified sugars, and modified internucleotide linkages. Morespecifically, the therapeutic oligonucleotides may be made up of variousnucleic acids, including natural and modified DNA, RNA, and derivativesthereof with modified bases, sugars, or linkages, of which somenon-limiting examples are PNA, LNA, and morpholino phosphorodiamidates.In one preferred embodiment, the therapeutic oligonucleotide targetedagainst the MYC oncogene may be comprised of peptide nucleic acid (PNA)residues, and linked to Ti-RGD (SEQ ID NO: 1) by a peptide sequencespecifically cleaved by matrix metalloproteinases, and a basic peptidesequence conducive to cellular uptake.

Angiogenesis is the growth of new blood vessels in the body and isnecessary for the repair or regeneration of tissue during wound healing.A lack of small blood vessel production can contribute to tissue deathin cardiac muscle after a heart attack. In cancers, angiogenesis enablescancers to enlarge and spread. The walls of blood vessels are formed byvascular endothelial cells and angiogenesis can stimulate them todivide. In cancer, two proteins appear to be the most important forsustaining tumor growth vascular endothelial growth factor (VEGF) andbasic fibroblast growth factor (bFGF). The binding of either VEGF orbFGF to its appropriate receptor on the cell activates a series of relayproteins that transmits a signal into the nucleus of the endothelialcells. The nuclear signal ultimately prompts a group of genes to makeproducts needed for new endothelial cell growth. Such signals could beused to trigger the release therapeutic molecules bonded to an implantsurface that could be used to alter angiogenesis of epithelial cellsadjacent or attached to the implant surface.

Other therapeutic molecules and peptides which can be bonded to thelinkers or organosilanes may be used to modify implant surfaces.Examples of such molecules include but are not limited tochemotherapeutics like Taxol [CAS 33069-62-4], camptothecans; kinaseinhibitors, such as erlotinib, flavopiridol, gefitinib, or imatinib;anti-rejection drugs that prevent restenosis like Sirolimus [CAS53123-88-9] and Tacrolimus [CAS 104987-11-3], cell growth factors,thrombolytic drugs, and osteoactive factors. Preferably the therapeuticmolecules and peptides have moieties which can be readily coupled to theorganic groups pendant from the silylated implant surface. Where suchgroups are unavailable, the therapeutic molecules and peptides may bechemically modified to provide a site for covalent bonding to theorganosilane or linker on the surface of the implant. For example, the17-hydroxyl of rifampin may be modified by esterification with2-methyl-4-thioethylamido maleic acid to form a sulfhydryl group. Thissulfhydryl group may be directly reacted with a silylated implantsurface having epoxide groups to form a thioether. Such modifiedtherapeutic molecules bonded to the surface of an implant may becharacterized for their therapeutic activity using methods disclosedherein. Where reactive groups present on a therapeutic molecule lead toimproper coupling with a silylated implant surface, protecting groupsmay be used to insure that only suitable groups like C-terminal carboxylgroups are available for coupling.

Therapeutic molecules, peptides, or therapeutic oligonucleotides may becovalently bonded using standard Fmoc coupling or other known methods tothe organosilane modified implant surfaces having terminal amino,sulfhydryl, or carboxy groups. For example, a five-fold molar excess ofFmoc-aminoethoxyethoxyacetic acid linker (AEEA, Applied Biosystems) canbe coupled using Fmoc reagents to silylated Ti—O—Si(CH₂)₃NH₂ substrates.The silylated substrates may be supported in a lab-made reaction columnmounted on a Rainin PS3 peptide synthesizer, or an Applied Biosystems430A peptide synthesizer, or an Expedite 8909 DNA/RNA/pNA synthesizerunder anhydrous conditions. Initially two AEEA linkers can be added; 3-5can be added if two linkers do not make the acid-labile linkersufficiently accessible. Following piperidine removal of the terminalFmoc protecting group, the N-terminal amine can be reacted with fiveequivalents of 2-methyl maleic anhydride in dry CH₃CN plus oneequivalent of dimethylaminopyridine (DMAP) for one hour. TheTi-substrates with a terminal methyl maleate can be washed withdimethylformamide, (CH₃)₂NCHO, then reacted with 10 equivalents of1,2-diaminoethine plus two equivalents of N,N′-diisopropylcarbodiimide(DPC) in CHCl₃ under anhydrous conditions. A five-fold molar excess ofvancomycin can be coupled to the resulting terminal amine using Fmocreagents to yield the desired stable acid-labileTi—O—Si(CH₂)₃NH-AEEA-AEEA-mVAN after a final wash with (CH₃)₂NCHO.

Using solid phase coupling, vancomycin or other therapeutic compound maybe covalently bonded to an oligo(ethylene glycol)-Ti surface. Forexample, a five-fold molar excess of AEEA can be Fmoc-coupled by solidphase methods to Ti-amino substrates under anhydrous conditions.Initially five AEEA linkers can be added; 6-10 can be added if fivelinkers do not make the vancomycin sufficiently accessible.Alternatively, a five-fold excess of N-Fmoc-amide-dPEG₄, MW 478.5available from Quanta Biodesign, Powell, Ohio, can be coupled to an APTSsilylated Ti surface to give Ti—O—Si(CH₂)₃NH-OEG. Following piperidineremoval of the terminal Fmoc protecting group, a five-fold molar excessof vancomycin can be coupled with Fmoc reagents to yield the desired(Ti—Si—OEG-VAN) Ti—O—Si(CH₂)₃NH-OEG-VAN as illustrated schematically inFIG. 6 after a final wash with (CH₃)₂NCHO.

A competitive ligand for vancomycin or other therapeutic molecule may beprepared by solid phase coupling of D-Ala-D-Ala to an oligo(ethyleneglycol)-Ti surface. For example, a five-fold molar excess ofaminoethoxyethoxyacetic acid t-butyl ester (AEEAtBu) can be Fmoc-coupledby solid phase methods to Ti-carboxy substrates under anhydrousconditions. Initially three AEEA linkers can be added; 4-8 can be addedif five linkers do not make the noncovalently bound vancomycinsufficiently accessible. Alternatively, N-Fmoc-amide-dPEG₄, MW 478.5available from Quanta Biodesign, Powell, Ohio, can be coupled to an APTSsilylated Ti surface to give Ti—O—Si(CH₂)₃NH-OEG. Following piperidineremoval of the terminal t-Bu protecting group, or terminal Fmocprotecting group, a five-fold molar excess of unprotectedα-amino-D-Ala-D-Ala-t-butyl ester can be coupled with Fmoc reagents toyield the (Ti—Si-Ala)Ti—O—Si(CH₂)₃NH-OEG-D-Ala-D-Ala after a final washwith (CH₃)₂NCHO. The loaded implant substrates can be washed twice withPBS, then saturated with a 10-fold molar excess of 1 mM vancomycin inPBS for 30 min. to yield the desired (Ti—Si-Ala-VAN),Ti—O—Si(CH₂)₃NH-OEG-D-Ala-D-Ala*(VAN) as shown schematically in FIG. 7followed by three washes with PBS to remove unbound vancomycin.

Chemical determination of the moieties tethered to the implant substratesurface can be determined by Matrix Assisted Laser DesorptionIonization-Time Of Flight (MALDI-TOF) mass spectroscopy.Characterization of the molecular ions resulting from the fragmentationof the modified vancomycin can be compared with values obtained fromunmodified vancomycin and published literature values to confirm thatlinkages to the metal and modification to the vancomycin occur.Derivatized Ti disks overlaid with sinapinic acid matrix can be allowedto outgas under high vacuum in the Ciphergen sample chamber for 30 min.before analysis. Molecular ion formation can be achieved through firingof a 338 nm laser onto the surface of the Ti implant, causing bondbreakage and release of the parent ion. Because of the innate charge ofthe molecular ions, the parent ion and any fragments can be acceleratedinto the mass spectrometer where the time of flight can be a function ofthe mass of the molecular ions. Computer assignment of the identities ofmolecular ions of Ti—Si-mVAN, Ti—Si—OEG-VAN, and Ti—Si—OEG-D-Ala-D-Alacan be based upon reported values for vancomycin and comparison with thepattern produced by underivatized vancomycin alone.

Detection of amount and density of acid-labile therapeutic moleculessuch as vancomycin bonded to a Ti implant may be determined byspectrofluorimetry and by epifluorescence microscopy. For example,tethered vancomycin can be incubated with a monoclonal antibody tovancomycin (US Biologicals, 1:100 dilution), 25° C., 60 min. inTris-buffered saline containing 1% goat serum, followed by reaction witha sheep anti-mouse FITC-coupled secondary antibody (Molecular Probes,1:80 dilution), 25° C., 60 min. Quantities can be estimated bycomparison with known amounts of vancomycin in the fmol-nmol range.Vancomycin density on the Ti surface can be determined byepifluorescence microscopy using a TRITC-inked secondary antibody to thevancomycin antibody. Digital images can be collected using a confocalscanning laser microscope to image titanium surfaces. The confocalityallows filtering out of the out-of-phase reflected light from the metalsurface.

Incubation of surfaces with weak acid may be used to characterize thestability and release of tethered or complexed bioactive or therapeuticmolecules bonded to an implant substrate surface. For example,Ti-vancomycin hybrids can be incubated in PBS (pH 5.0-8.0) for 1-180min. The PBS pH can be adjusted using the H₃PO₄:Na₂HPO₄ ratio, whileholding ionic strength constant. The most “active” pH values, which maybe pH 5.0, 5.5, and 6.0, as well as physiological pH 7.4 may beevaluated in a time course where antibiotic release can be measured at1, 2, 3, and 24 h.

The activity of the surface modified implant substrates with covalentlyattached, labile or complexed therapeutic molecules may be measured byincubating them in the presence of the cells that the molecules arechosen to interact with. For example, the bactericidal activity oftethered vancomycin against S. aureus infections may be measured. Thebactericidal activity of these surfaces may be tested against an initialinoculum of 10³ to about 10⁶ S. aureus, a concentration that can occupyless than 1% of a sample 177 mm² modified Ti disk. Control surfaces cantest the proliferation of the bacteria on the Ti alone, the effect ofsoluble vancomycin on bacterial propagation, the effect of the linkeralone, and finally the effect of irreversibly immobilized vancomycinwithout OEG on bacterial proliferation. The proliferation curve of thebacteria can be determined every 60 minutes for 4 h by extraction of theDNA and measurement using PCR. Numbers of bacteria can be estimated bycomparison with PCR signals using DNA isolated from known numbers ofbacteria. In addition, pH measurements can be performed to determine ifbulk changes in pH are occurring as a result of bacterial propagation.Finally, bacterial number can be measured by direct counting afterplating serial dilutions on agar plates. Colony formation can beassessed by cell counting after photography and total numbers ofcolonies calculated. These values can be compared with the numbersdetermined by PCR. A Molecular Probes “Live/Dead BacLight” kit may alsobe used to visualize bacterial viability based on differential staining.

Implant substrates or portions thereof that are modified or unmodifiedcan be sterilized by exposure to UV light and placed into the well of a24-well tissue culture plate. 0.5 mL of DMEM containing 10% FBS can beadded and medium can be inoculated with 10³-10⁶ infectious units of S.aureus. The inoculum can be mixed by agitation on a rotary shaker for 5minutes, followed by incubation at 37° C. under normal tissue cultureconditions, i.e., in a 5% CO₂, humidified incubator with no agitation.Bacteria proliferation can be measured as a function of time (1-24 h).

Isolation of DNA from adherent and non-adherent bacteria. Non-adherentbacteria can be gathered in the supernatant, pelleted, and resuspendedin 100 μL PBS. Adherent bacteria on the surface can be bathed in 100 μLPBS. Cell lysis can be achieved by four freeze/thaw cycles, followed bycentrifugation. The supernatants can be used for determination ofbacterial number by PCR.

PCR may be used to determine bacterial number. For example, 10-20 μL ofthe above DNA solutions can be used in a 100 μL reaction volume using aTris-KCl—MgCl₂ buffer and AmpliTaq, as described in the paper. Primersare directed at the 16S rRNA (Forward: CGGCAGGCCTAACACATGCAAGTCG.Reverse: GGTTGCGGCCGTACTCCCCAGG) to yield an 881 bp product, after 30-35cycles. Detection can be by ethidium bromide or SybrGreen II stainingafter fractionation by agarose gel electrophoresis.

Determination of S. aureus numbers by dilution. Supernatant can beremoved from cultures containing S. aureus as above and the chemicallymodified surfaces can be washed with 2 mL of DMEM and pooled with thesupernatant from the same culture. Following agitation, this supernatantcan be plated on LB agar plates at dilutions of 0, 10², 10⁴, 10⁶ fordetermination of bacterial numbers. Additional dilutions can be used, asappropriate, to yield a countable number of colonies on the plate.Surfaces can be photographed and cell numbers determined by direct cellcounting. Determinations can be performed by two or more independentinvestigators.

Visualization of adherent and floating viable and non-viable bacteria.Using the Live/Dead BacLight system (Molecular Probes), live and deadbacteria can be stained with SYT09+propidium iodide. Live cells canfluoresce green, whereas cells with compromised membranes can fluorescered. Digital images can be recorded of surface adhered and floatingbacteria and ratios of live:dead bacteria determined in random fieldsusing double immunofluorescence.

The activity of various therapeutic molecules and their biocompatibilitymay be determined by measuring the proliferation and maturation oftarget cell on modified implant surfaces. For example, the bactericidalactivity and biocompatibility of tethered vancomycin of the presentinvention can be determined in the presence of cells. The effect on theproliferation and maturation of chondrocytes and of osteoblasts, thecell types important for fracture healing can be measured. Importantly,the surface as designed, should be stable to both chondrogenic andosteoblastic cell proliferation, i.e., preferably release antibiotic, asappropriate, in the presence of bacteria. Using two concentrations ofbacteria that were killed when incubated with the active surface, theeffects of a culture with ATDC5 pre-chondrogenic and MC3T3-Elosteoblast-like cells can be determined. These cells can be plated at adensity of 1×10⁴ cells/1.77 cm² implant substrate at the same time asinoculation of bacteria.

For the acid-labile linkage, acidic solutions can cause significant, andtime-dependent release of a therapeutic tethered molecule likevancomycin. In the neutral pH range, there may be extremely slow orminimal release of the therapeutic molecule from the implant. For thecovalent linkages, such as OEG-VAN, and complexed bioactive moleculesuch as Ala surfaces, the linkages should be pH stable. Should theacid-labile linkage cause an immediate release of antibiotic, thelinkage may be modified so as to decrease its acid lability. This may bedone by decreasing the electron density at the cleavage site making itless likely to be protonated. Preferably the site of modification of thetherapeutic molecule is such that the activity of the molecule is notreduced. The active sites of the molecules would be known to thoseskilled in the art.

The amount and number density of therapeutic molecules tethered to theimplant surface that are required to produce an effect on cells near thesubstrate can be determined by measurement of the numbers of adherentand planktonic bacteria or other cells via PCR and colony formationassays. The live to dead ratio of cells on the surface of surfacemodified substrates and in solution using dyes specific for living ordead cells such as bacteria may also be used. Should surface boundtherapeutic molecules be insufficient to interact with cells and cellcolonies near the implant surface, multivalent linkages may be used toincrease their surface concentration.

Embodiments of the present invention include surfaces with importantclinical applications. The first surfaces include therapeutic moleculestethered or associated with an implant surface through a ligand orlinker connecting the therapeutic molecules and implant surface.Additional modifications are possible to further develop these surfaces,as are the addition of other site-directed delivery systems.

Testing of the covalently modified or derivatized surfaces applied tosubstrates such as an intramedullary nail in an animal models can beused to establish whether the derivatized implants maintain theirtherapeutic (chemotherapeutic, anti-inflammatory, proliferative, orbactericidal) activity in vivo and act to heal fractures or performsensing functions. Substrates, such as but not limited to a layer oftethered therapeutic molecules on an implantable substrate may beevaluated using animal testing. Without limitation and for illustrativepurposes only, suitable therapeutic molecules may include an antibiotic,and the implantable substrate an intramedullary nail.

Quantitative microCT can be used to assess the structural properties ofthe developing fracture callus and the formation of trabeculae in thehealing cancellous bone. Micro-tomographic imaging values are inexcellent agreement with the indices assessed from conventionalhistomorphometry and are an excellent predictor of bone quality andstrength. In vivo microCT analysis may be used to dynamically monitorthe healing of the bone, its structure, and its mechanical strength,over the course of animal experiments. An infected fracture cannot healproperly and the microCT can be used to provide an indication of anestablishing infection and the performance of a surface modifiedsubstrate.

Covalently modified intramedullary nails can be implanted into thefemurs of Wistar Rats and the femurs fractured using an Einhornprotocol. After confirmation of fracture by radiology, an infectiousagent such as S. aureus can be injected into the site of the fracture.To prevent sepsis, the concentration of S. aureus can be kept very low(1-50 cfu). Ten rats can comprise a sample group, with a treated nailinserted on one side and an untreated nail inserted on the contralateralcontrol side. Using this general testing methodology, it is alsopossible to evaluate the effects of an implant surface modified bycovalent attachment of linkers and therapeutic molecules. The fracturesites can be monitored at 7, 14 and 21 days. Animals can be sacrificedat these time points and fracture healing assessed by conventionalhistology and morphometry. Evaluation of the systemic infection andserum antibiotic levels may be determined at these time periods. MicroCTcan also be used to monitor fracture healing at 0, 7, 14 and 21 days, aswell as to obtain information on the mechanical strength of the bone. Atthe termination of the experiment, bone can be X-rayed, harvested andits mechanical properties measured.

Skeletally mature, male, Wistar rats, weighing 300-500 grams, can beanesthetized by intraperitoneal injection of ketamine (20-60 mg/kg) andxylazine (2.5 mg/kg) anesthesia. Hind limbs can then be shaved, washedwith Betadine (Povidine-Iodine), and under aseptic conditions, a medialparapatellar arthrotomy made and the patella dislocated laterally. Theintramedullary canal can be entered through the intercondylar notch andreamed with an 18-gauge needle. Modified or unmodified intramedullarynails can be prepared from 1.14×26 mm Kirschner wire (Smith and NephewRichard, Inc., Memphis, Term., USA). The nail can be inserted into thecanal, the patella relocated, soft tissues closed with 4.0 nylonsutures, and the skin closed with staples. This stabilized femur can beplaced in a three-point bending device and the femur fractured by theforce of a 500-gram weight dropped 35 cm. Bilateral fractures can bemade in all animals. X-ray analysis can be used to confirm the positionand orientation of each fracture. Fractures outside the central 8 mm ofdiaphysis, and oblique or spiral fractures, can be excluded from thestudy. After the animals are killed, the femora can be disarticulatedand radiographs made for assessment of fracture-healing. Each fracturespecimen can be reviewed and classified for callus maturity according tothe classification wherein stage 1 indicates nonunion; stage 2, possibleunion; and stage 3, radiographic union.

After confirmation of the fracture by microCT, an aliquot containingabout 10⁴ to about 10⁶ cfu of S. aureus in 100 μL can be injected intothe medullary cavity.

All nails can be fitted snugly and no other fixation usedpostoperatively. Rats can be allowed unrestricted ambulation in theircages after recovery from anesthesia, and can be observed daily foractivity and weight bearing as indicators of adequate fractureimmobilization and freedom from pain. All femora can be imaged again atthe time of sacrifice, with the contralateral femora serving as control.At 7, 14, and 21 days, i.e., the time of sacrifice, blood samples can becollected for determination of blood levels of bacteria and antibiotics.Harvested tissues can also be assessed for bacterial infection. At 7, 14and 21 days, aliquots of blood taken from the test mammals can beevaluated for antibiotic load and the presence of S. aureus.

Femora from five animals from each sample can be utilized forhistological and morphometric analysis, as known to those skilled in theart. Immediately after animal sacrifice, fracture calluses can beremoved, fixed for two to three days in 10 percent neutral bufferedformalin followed by two days in Bouin's solution and then decalcifiedin a 10 percent acetic acid, 0.85 percent NaCl, and 10 percent formalinsolution. Since the nail cannot be integrated, it can also be retrievedaseptically for testing of antibiotic load, infection status and surfacecharacteristics. Specimens can be embedded in paraffin, sectionedlongitudinally, and stained with hematoxylin and eosin or Masson'strichrome. The progression of fracture-healing in each specimen can bemeasured with use of a scale that assigns a grade based on the relativepercentages of fibrous tissue, cartilage, woven bone, and mature bone inthe callus. Grade I indicates fibrous tissue; grade 2, predominantlyfibrous tissue with some cartilage; grade 3, equal amounts of fibroustissue and cartilage; grade 4, all cartilage; grade 5, predominantlycartilage with some woven bone; grade 6, equal amounts of cartilage andwoven bone; grade 7, predominantly woven bone with some cartilage; grade8, entirely woven bone; grade 9, woven bone and some mature bone; andgrade 10, lamellar (mature) bone. Four slides can be examined for eachfracture.

Histomorphometric evaluation of trabecular structure and chondrocytecharacteristics can be carried out with an Osteo Metric's image-analysissoftware system (Atlanta, Ga.). Measurements of trabecular structure andof the size and number (cell density) of hypertrophic chondrocytes canbe made in the interface region (the region beneath the subperiostealnew bone formed by intramembranous ossification and the ends of thefractured femur). Measurements of the hypertrophic chondrocytes can bemade in the cartilage within about 258.5 micrometers of this interface.Parameters that can be evaluated include cell size (in squaremillimeters) and cell density (in cells per square millimeter).Evaluation of endochondral bone included (1) the area occupied bytrabeculae composed of spicules of calcified cartilage covered byosteoid and newly formed bone, (2) the thickness of these trabeculae,and (3) the percentages of bone and calcified cartilage in thesetrabeculae.

For the microCT imaging, the unprocessed fractured bone of the livingrats can be evaluated at 0, 7, 14, and 21 days using a compactfan-beam-type tomograph (Scanco Medical AG, Bassersdorf, Switzerland). Atypical examination can consist of a scout view, selection of theexamination volume, automatic positioning, measurement, offlinereconstruction, and evaluation. For each sample, a total of 286micro-tomographic slices with a slice increment of 14 μm can be acquiredcovering 4 mm of the total length of the fractured bone. The totalexamination time per rat limb can be approximately 30 min for a voxelsize of 14 cm³. For the subsequent analysis, a subvolume of theoriginally measured data can be selected. This selected volume ofinterest (VOI) can be located in the center of the fracture site. Thesize of the VOI can be set at 4 mm³ (286×286×286 voxels). In the nextstep, bone tissue can be separated from marrow using a thresholdingprocedure. All samples can be binarized using the same parameters forthe filter width, the filter support, and the threshold (about 10.2% ofmaximal possible gray value). For the 3D visualization an extendedmarching cubes algorithm may be used. At the completion of the study,and after animal sacrifice, the healing bone can be evaluated by theconventional techniques.

The efficacy of a tethered bioactive molecule such as vancomycincovalently bonded on an implant to minimize biofilm formation may bedetermined by recovery of the implant at the end of the animal study andits incubation in an LB broth. Serial dilution techniques may be used todetermine the number of bacteria that remain on the implant surface. Thecontralateral untreated nail can be used as a control. Because theinfection is systemic, both nails should be equally exposed to thepresence of bacteria.

The stability of a tethered therapeutic molecule such as vancomycincovalently bonded to an implant surface to the in vivo conditions can bedetermined after retrieval of the surface-modified implant nails. Theretrieved, derivatized or modified implant can be washed extensively andthen for example reacted with the antibody to vancomycin. Reaction witha FITC-labeled secondary antibody and imaging by confocal laser scanningmicrocopy can give a rough estimate of the density of the antibodyremaining after the in vivo incubation. As controls, modified butunimplanted substrate nails can also be visualized to determinevancomycin surface density.

Specimens can be kept moist, wrapped in saline saturated gauze, andstored in air tight containers at 4° C. until day of testing. On the dayof testing, femora can be kept moist and brought up to room temperature,mounted in bone cement to leave a 15 mm length of diaphysis, andsubjected to mechanical testing.

Each femur may be subjected to torsion at 15°/min until failure using ajig that ensures proper alignment and that is designed to converttranslational displacement to angular displacement using an Instronhydraulic materials testing machine. Load-displacement curves may becontinuously recorded on a plotter, digitized using a flat-bed scanner,and analyzed using Scion Image, beta version 3b (Scion Corporation,Frederick, Md.). Ultimate torque (N−m), stiffness (N−m/deg) (slope ofload-deformation curve measured within proportional range), angle ofdeformation to failure (deg), and energy absorption to failure (N−m×deg)(area under the load-deformation curve) can be calculated. Gross failurepatterns can be detected visually and can be classified as (I) failurethrough the fracture site and adjacent host bone, or (II) failureindependent of the fracture (i.e. through host bone only).

All data can be analyzed by one-way and two-way ANOVAs to teststatistical significance (p<0.05) between groups and time points.Multiple, planned comparisons between groups and time points can beperformed using orthogonal contrasts (SYSTAT, Systat, Inc., Evanston,Ill.).

Correlations can be performed to study the interdependence/associationbetween temporal changes in mechanical properties of the long bones andhistomorphometric parameters at the fracture site. Pearsonproduct-moment correlation coefficients, r, can be determined for eachmechanical property versus each histomorphometric parameter over time.The average of each variable per experimental group per time point canbe used. The null hypothesis, no correlation, can be rejected for asignificance level of 5%.

For implants modified with therapeutic molecules to prevent bacterialinfections, restenosis in implanted stents, tumor recurrence in apreviously treated tumor, fouling of a sensor, or other cellularconditions, the outcome of animal study tests would be surfaces free ofor having substantially reduced numbers of the undesirable cells. In thecase of an implant to repair fractured bone or dentin, the outcome wouldbe healing of the fracture without the presence of undesirable cells onthe implant surface. In particular for intramedullary implants modifiedwith antibiotics, the outcome should be infection-free, fracturehealing, normal bone structure as assessed by microCT and histology, andbiocompatibility as assessed by histology.

The first measure of the outcome can be in the infection-free healing ofthe fracture. The bacteria injected into the site of fracture willdisrupt normal bone healing and may be used to monitor whetherparticular linkers and therapeutic molecules covalently bonded to theimplant surface are effective. If a significant infection is present inthe bony cavity, healing can be retarded and non-union can be the likelyresult. With respect to infection, therefore, the assessment of thedegree of bone healing relative to that in bacteria-free controls is ameasure of the effectiveness of a surface modification orderivatization. For example, the test result for the presence of an S.aureus infection directly upon harvest of the bone can be used todirectly measure if the presence of bacteria affects the bone healing aswell as if the surface modification is successful in eliminatinginfection by day 21. Bone structure can be assessed during healing byuse of microCT as well as histology. Histological evaluations can occuron a subset of the animals throughout the healing period as well as onthe remaining animals at the end of the study. The histology data can becorrelated with the microCT data to assess the relative differences intrabecular structure, callus formation, mechanical strength, andbacterial load as a function of the antibiotic and the linkagecovalently bonded to the implant surface. Specifically, for fracturesthrough the aid of the microCT, the tightness of the bone interface withthe nail may be determined. In cases of infection and fracture amodification on the implant surface that is not effective is expect toshow an increasing space around the nail, poor healing, and soft tissuedamage. In the case of an implanted sensor, the sensitivity of thesensor over the course of the animal study wherein the animal isinjected with a trial of an agent may be used to assess the activity andfunction of a modified implanted senor surface with linkers andtherapeutic molecules designed to eliminate fouling by undesirable cellgrowth on the sensor. Embodiments of the present invention may befurther understood by reference to the following non-limiting examples.

EXAMPLE 1

This example illustrates that implants may be derivatized by covalentlybonding bioactive peptides to their surfaces.

Aminopropyl triethoxy silane (APTS) linkers can be used that allowreactions of peptides or molecules containing peptide-like moieties.Specifically, RGD (SEQ ID NO: 1) peptides were covalently bonded to asilicon wafer using APTS as a derivatizing agent. The presence of RGD(SEQ ID NO: 1) on the surface was determined by time-of-flight secondaryion mass spectrometry and surface roughness was measured by AFM. TheAPTS alone caused a larger increase in roughness than reaction withAPTS-linked RGD (SEQ ID NO: 1), probably the result of multiple layersof APTS. Inclusion of (CH₃)₂NCHO washes and sonication after thesilanization step ensures that only covalently bonded ATPS organosilaneremain on the surface, with the unbound APTS removed. This experimentdemonstrates that RGD (SEQ ID NO: 1) is directly linked to the siliconsurface via the APTS linker.

Osteoblasts were bound to the modified surface. Spinning disc technologymay be used to measure cell adhesive strength as a function of theapplied centripetal force. Adhesive strength of the osteoblasts wasmeasured by calculating the shear stress on the cells (speed of thespinning disc as a function of distance from the center of the disc).Covalently-bound RGD (SEQ ID NO: 1) increased adhesive strengthsignificantly over physisorbed, RGD (SEQ ID NO: 1), APTS-modifiedsilicon and silicon alone. Osteoblasts, plated on these RDG surfacesshowed a well-spread morphology, characterized by large, patchy focaladhesion contacts and increased integrin expression; osteoblasts platedon control surfaces were significantly less well-spread. Furthermore,inhibition of osteoblastic attachment by integrin antibodies inhibitedcellular attachment.

Without wishing to be bound by theory, it may be that adhesion ofosteoblasts to an RGD (SEQ ID NO: 1)-engineered surface stimulates theirmaturation and mineralization. As assessed by RT-PCR, expression ofphenotypic markers of osteoblast maturation was increased in cellsplated on the modified surfaces. Furthermore, alkaline phosphatase andalizarin staining was also increased. FTIR analysis showed that thedeposited mineral was biological apatite.

EXAMPLE 2

This example shows how a therapeutic molecule that is an antibiotic maybe immobilized by covalent bonding to an implant surface. An antibioticcan be immobilized on a Ti surface and that the tethered antibioticmaintains its antibacterial activity. Vancomycin can be used as arepresentative antibiotic.

For this purpose, a 4.37 g sample of 350 mesh Ti6Al4V particles werecleaned with 5 mL of 50% MeOH/50% conc. HCl for 20 min. at roomtemperature, with vortexing for 20 sec. at 0, 5, 12, 15, and 20 min. Theparticles were washed twice with 5 mL of double-deionized water, thenfour times with 5 mL of anhydrous dimethylformamide [(CH₃)₂NCHO]. Afterremoving remaining (CH₃)₂NCHO supernatant, the particles were driedovernight under vacuum in the entry chamber of a Vacuum AtmosphereMO-20M glove box. Next morning, the particles were taken into the argonatmosphere chamber of the glove box and washed twice with 10 mL ofanhydrous toluene, resuspending them with a stainless steel spatula.Then the particles were modified with 5 mL of 5% (v/v) APTS in anhydroustoluene for 60 min., stirring the particles with a stainless steelspatula at 0, 10, 30, and 60 min. The particles were then washed twicewith 10 mL of anhydrous (CH₃)₂NCHO, resuspending them with a stainlesssteel spatula. The supernatant was removed and the particles were driedunder vacuum overnight in the entry chamber of the glove box.

Next morning, the particles were removed from the entry chamber of theglove box and a 0.1168 g sample was assayed for amine content. Thesample was resuspended in 1 mL of 0.35 mM SnCl₂, 0.1 M Na citrate, pH 5,then mixed with 1 mL of 4% (w/v) ninhydrin in EtOH. The suspension wasimmersed in a boiling water bath for 15 min., cooled for 2 min., thendiluted with 5 mL of 60% EtOH/40% H₂O. One mL of the 7 mL supernatantwas diluted with 9 mL of 60% EtOH/40% H₂O. The 10-fold dilution yieldedA₅₇₀=0.324. Using ε₅₇₀=1.5×10⁴/M cm, calculated=21.6 μM, whichcorresponded to aminopropyl derivatization of 13.0 μmol/g for themodified Ti6Al4V, lot 030712-01. The aminopropyl-modified particles ofTi6Al4V will provide the support for Fmoc coupling of linkers, unusualamino acids, and vancomycin.

A Glen Research solid phase synthesis column was charged with 1.29 g ofaminopropyl-Ti6Al4V, lot 030712-01, and inserted into position 2 of aRainin PS3 peptide synthesizer as shown in FIG. 1. The support waswashed twice with 5 mL of anhydrous (CH₃)₂NCHO, then automaticallycoupled with a four-fold molar excess of Fmoc-aminoethoxyethoxyaceticacid linker activated with a four-fold molar excess ofN-[(dimethylamino)-1-H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide, followed by automatic Fmoc deprotectionwith 20% piperdine/80% (CH₃)₂NCHO. The deprotection solution and washwere collected for ultraviolet absorbance spectroscopy. A second AEEAlinker was coupled, and the second Fmoc deprotection solution and washwere collected for analysis. Similarly, a four-fold excess of vancomycinwas coupled automatically by the same protocol.

After the third coupling, the column was washed twice with 5 mL ofanhydrous (CH₃)₂NCHO, then dried under vacuum overnight. Absorbancespectra of the deprotection solutions revealed 301 nm peakscharacteristic of the 9-piperidino-dibenzofulvene product of Fmocdeprotection. Using ε₃₀₁=7.78×10³/M cm, it was calculated thatquantitative addition of the two AEEA linkers to the aminopropyl-Ti6Al4Vsupport occurred. The VAN-AEEA-AEEA-NH—Pr—Ti6Al4V preparation (Van-Ti),lot 030718-01, can be analyzed by MALDI-TOF mass spectroscopy, thentested for its ability to bind peptidoglycan and to inhibit growth of S.aureus.

Chemical determination of the moieties tethered to the Ti particles wasanalyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight(MALDI-TOF) mass spectroscopy. Derivatized Ti particles overlaid with a2,5-dihydroxy-benzoic acid matrix were allowed to outgas under highvacuum in the Ciphergen sample chamber for 30 min. before analysis.Molecular ion formation was achieved through firing of a 338 nm laseronto the surface of the Ti, inducing bond breakage and release of aspectrum of product ions, which were accelerated into the massspectrometer by a strong magnetic field. The mass spectrum observed isshown in FIG. 2. The time of flight in this situation is a function ofthe mass of the molecular ions. In this first attempt to sputter VANfragments from the Ti surface, many small peaks appeared. Assignment ofthe identities of molecular ions of the putativeVAN-AEEA-AEEA-NH—Pr—Ti6Al4V were attempted based on the known masses ofvancomycin, 1449 Da, and the AEEA linker, 163 Da. Reasoning from thestructure, the 1537 Da peak might be VAN-NH-EtOEtOH, the 1472 Da peakmight be a Na⁺ VAN, and the 1402 Da Peak might be decarboxylated VAN.

Lys-D-[¹⁴C]Ala-D-[¹⁴C]Ala soluble peptidoglycan binding to VAN-Ti. Anempty polypropylene vial and 100 mg samples of Ti6Al4V,aminopropyl-Ti6Al4V, lot 030712-01, and VAN-AEEA-AEEA-NH—Pr—Ti6Al4V, lot030718-01, were resuspended in 1 mL of PBS, and rocked for 1 hr at roomtemperature. The supernatants were removed and replaced with 2.5 nCi ofLys-D-[¹⁴C]Ala-D-[¹⁴C]Ala soluble peptidoglycan, and then rocked for 2hr. The supernatants were removed and counted in 10 mL of Aquasol II(PerkinElmer Life Sciences) in a Beckman LS 6000SC scintillationspectrometer. The VAN-Ti preparation bound more of the labeledpeptidoglycan (50-60%) than aminopropyl-Ti6Al4V or unmodified Ti6Al4V(15%). To reduce the potential for nonspecific binding, the experimentwas repeated, replacing the PBS in the washing step with 0.1% (w/v)bovine serum albumin in TBST. The samples were rocked at roomtemperature for 4 hours and overnight at 4° C. After three washes withPBS to remove unbound label, the supernatants were removed and counted.Binding was reduced in each sample subsequent to the albumin blockingstep. The vancomycin-containing derivative bound 25% of the label,whereas the other two derivatives bound 5% or less.

EXAMPLE 3

This example illustrates the bactericidal activity of a therapeuticantibiotic molecule vancomycin bonded to an implant surface, VAN-Ti,against S. aureus infections.

To assess the bactericidal activity of the Ti-grafted antibiotic, thefollowing experiment was performed. Ten μL of an overnight culture of S.aureus was inoculated into 2 mL of LB/1% dextrose, incubated for 2 h at37° C. with vigorous aeration, pelleted, resuspended in 1 mL and 10 μLof that culture used to inoculate wells containing 200 μL PBS/1%dextrose and APTS-derivatized Ti, VAN-Ti, or PBS alone; modified Tisamples had been subjected to three PBS washes prior to use. Sampleswere incubated for 1 h at 37° C., washed with PBS, and stained with theLive/Dead® BacLight™ Bacterial Viability Kit (Molecular Probes). Liveand dead bacteria were visualized by confocal microscopy. The results ofrepresentative fields resulting from this treatment are presented inFIG. 3. S. aureus incubated on Ti that has been derivatized with thelinker APTS are shown in the top row (A) and (B). Most bacteria staingreen (light gray), with only dead cells exhibiting a red fluorescence(light gray). Many live bacteria are evident, with a small amount of redstaining as would be expected in any culture of bacteria. The bottomrow, (C) and (D) shows S. aureus that were incubated on thevancomycin-tethered surface (VAN-OEG-APTS-Ti) Note the reduction in thelawn of live bacteria, predominantly black (C) and a concomitantincrease in the numbers of dead bacteria by red fluorescence (light gray(D)). The results clearly indicate that the vancomycin derivatized Tisurface retains its antibiotic activity and kills the adherent S.aureus. The results show that an active antibacterial surface wascreated on an implant and that such implants are useful in treatment orprophylaxis of periprosthetic infection.

EXAMPLE 4

This prophetic example demonstrates preparation and characterization ofa surface with therapeutic molecules covalently bonded to an implantsurface that enhances bone cell attachment and thereby facilitatestissue-implant integration, while remaining a long-term reservoir ofantibiotics to eradicate any infections that arise from hematogenoussources.

The implant can include covalently linked RGD (SEQ ID NO: 1)-containingpeptides to a Ti, creating a peptide-Ti hybrid. This can provide a mixedsurface as some of the RGD (SEQ ID NO: 1) motifs will serve as part ofthe linker for the immobilization of the therapeutic moleculetigecyclin. The RGD (SEQ ID NO: 1)-tigecyclin linkage will be labile,thereby exposing additional RGDs (SEQ ID NO: 1) for promotion ofosseointegration as the antibiotic is released.

For enhanced osseointegration, the RGD (SEQ ID NO: 1) peptide motif canbe used; it is one of the most common cell binding ligands; and it iscontained within the major bone proteins (e.g., collagen type I,osteopontin and bone sialoprotein). In addition to providing animmediate connection between the cell and the surface, the interactionbetween RGD (SEQ ID NO: 1)-containing peptides and integrin receptorsplay a significant role in the regulation of osteoblast function andbone development. In bone, these markers include elevation of alkalinephosphatase activity, increased osteocalcin, osteopontin and type Icollagen expression, and evidence of mineral deposition. The bindingactivity of the RGD (SEQ ID NO: 1) motif is modulated by flanking aminosequences. For example, the loop region of the attachment portion offibronectin includes both RGD (SEQ ID NO: 1) as well as flanking aminoacids such as but not limited to (PGVDYTITVYAVTGRGDSPASSKPVSINYR) (SEQID NO: 6). The effect of such flanking amino acid sequences may bedetermined by progressively increasing the number and type of flankingamino acids at each side of the RGD (SEQ ID NO: 1) motif and anchor onthe modified peptides covalently bonded to the Ti disc.

Cells will be plated on RGD (SEQ ID NO: 1)-modified surfaces and cellbinding and differentiation will be evaluated. Cell binding will beevaluated using both cell attachment assays and the spinning discmethodology, while differentiation will be evaluated using RT-PCRevaluation of osteoblast phenotypic markers, as well as measurement ofalkaline phosphatase activity and mineral deposition. Controls for thestudy will include motifs such as RGE, RGES and scrambled peptides, aswell as unmodified Ti and linker-modified Ti.

The broad spectrum, new generation antibiotic tigecyclin will betethered to the metal on a background of this adhesion peptide to yielda surface that is both osteogenic and bactericidal. Tigecyclin may belinked to Ti-RGD (SEQ ID NO: 1), as illustrated in FIG. 9 (A), byacid-labile maleamide or hydrazone linkages as shown in FIG. 8. Bothmethylmaleamide and hydrazone linkages display stability atphysiological pH, but are readily hydrolyzed in mild acid, pH 5-6. Inthe protected environment surrounding a Ti implant, bacterialproliferation leads to areas of lowered pH (in solution, this drop is >4pH units). These linkers will be cleaved under mildly acidic conditions(pH 5.0-6.0), releasing tigecyclin into the bacterial slime where itwill kill the adherent microorganisms. For tigecyclin to be active, itmust be released from the Ti surface and taken up by bacteria. Releaseof tigecyclin from the Ti surface will be triggered by the acidicbacterial microenvironment. The released antibiotic will then targetthese bacteria. Accordingly, the infection is eliminated at its sourcein a time- and site-specific manner. Cleavage of the acid-labile linkersto release the antibiotic will expose the RGD (SEQ ID NO: 1) motif asillustrated FIG. 9(B).

The modified Ti surfaces can be evaluated for antibiotic stabilityand/or release kinetics under acidic conditions and for bactericidalactivity against adherent S. Aureus. The modified surface may also becharacterized to determine if the modified surface supports osteoblastproliferation and maturation both in the presence and absence of alow-level bacterial infection.

Ti alloy (Ti6Al4V) will be purchased from Stryker Howmedica Osteonics(Allendale, N.J.) as highly polished disks identical to those used insurgical practice. Surfaces will be cleaned with HCl/MeOH prior to useand will be evaluated for surface smoothness by SEM.

Modification of the Ti surface. RGD (SEQ ID NO: 1) peptides will becovalently attached to an activated, smooth Ti surface using silanechemistry. Briefly, discs will be washed with anhydrous toluene, thentreated with 1.0-5.0 mM 3-aminopropyltriethoxysilane (APTS) in toluenefor 45-90 min. The discs will then be sonicated in toluene followed bydimethylformamide, (CH₃)₂NCHO, to remove excess reactants. The aminodiscs will provide the base for Fmoc coupling of linkers, unusual aminoacids, and tigecyclin.

Solid phase coupling of acid-labile Tigecyclin to the Ti surface. Afive-fold molar excess of Fmoc-aminoethoxyethoxyacetic acid linker(AEEA, Applied Biosystems) will be coupled using Fmoc reagents toTi-amino discs supported in a lab-made reaction column mounted on aRainin PS3 peptide synthesizer under anhydrous conditions. The RGD (SEQID NO: 1) or RGDS (SEQ ID NO: 2) sequence, or more extensive sequences,or control sequences, will be assembled from the N-terminus of thelinker by normal Fmoc coupling. The acid-labile tigecyclin derivativeswill be added at only 0.5 equivalents in order to assure that at least50% of the RGD (SEQ ID NO: 1) or RGDS (SEQ ID NO: 2) peptides are freefrom the outset, as illustrated in FIG. 10 (A).

Following piperidine removal of the last amino acid Fmoc protectinggroup, the N-terminal amine will be reacted with 0.5 equivalents of2-methyl maleic anhydride in anhydrous (CH₃)₂NCHO plus one equivalent ofdimethylaminopyridine (DMAP) for one hour. The excess unreactedN-termini will then be capped with acetic anhydride. The Ti-discs with aterminal methyl maleate will be washed with (CH₃)₂NCHO, then reactedwith one equivalent of 1,2-diaminoethane plus two equivalents ofN,N′-diisopropylcarbodiimide (DPC) in (CH₃)₂NCHO under anhydrousconditions for 2 hr, followed by 2 washes with (CH₃)₂NCHO. Twoequivalents of tigecyclin in the carboxylate form in (CH₃)₂NCHO will becoupled to the resulting terminal amine using Fmoc activator HATU toyield the desired acid-labile Ti—Si—RGDS (SEQ ID NO: 2)-mTIG after afinal wash with (CH₃)₂NCHO.

Following piperidine removal of the last amino acid Fmoc protectinggroup, the N-terminal amine will be reacted with 0.5 equivalents ofsuccinic anhydride in anhydrous (CH₃)₂NCHO plus one equivalent ofdimethylaminopyridine (DMAP) for one hour. The excess unreactedN-termini will then be capped with acetic anhydride. The Ti-discs with asuccinate will be washed with (CH₃)₂NCHO, then reacted with oneequivalent of t-butyl carbazate (Boc-NHNH₂) plus two equivalents ofN,N′-diisopropylcarbodiimide (DPC) in (CH₃)₂NCHO under anhydrousconditions for 2 hr, followed by 2 washes with (CH₃)₂NCHO. The Bocprotecting group will be decomposed with 20% CF₃CO₂H in (CH₃)₂NCHO. Theresulting hydrazide will then be coupled with two equivalents oftigecyclin in the ethyl ketone form in (CH₃)₂NCHO for 24 hr to yield thedesired acid-labile Ti—Si—RGDS (SEQ ID NO: 2)-hTIG after a final washwith (CH₃)₂NCHO.

MALDI-TOF Mass Spectroscopy. Chemical determination of the moietiestethered to the Ti surface will be determined by Matrix Assisted LaserDesorption Ionization-Time Of Flight (ALDI-TOF) mass spectroscopy ofproducts removed from the discs with aqueous 0.1% CF₃CO₂H, pH 2.Characterization of the molecular ions resulting from the fragmentationof the modified Tigecyclin will be compared with values obtained fromunmodified Tigecyclin and published literature values to confirm thatlinkages to the metal and modification to the Tigecyclin occur asdesigned. To characterize peptides remaining on the acid-washed Tidiscs, they will be overlaid with a sinapinic acid matrix and allowed tooutgas under high vacuum in the Ciphergen sample chamber for 30 min.before analysis. Molecular ion formation will be achieved through firingof a 338 nm laser onto the surface of the Ti, causing bond breakage andrelease of the parent ion. Because of the innate charge of the molecularions, the parent ion and any fragments will be accelerated into the massspectrometer where the time of flight will be a function of the mass ofthe molecular ions. Computer assignment of the identities of molecularions of Tigecyclin and RGDS (SEQ ID NO: 2) peptides will be based uponreported values for Tigecyclin and comparison with the pattern producedby underivatized Tigecyclin alone.

The surface of the modified Ti will be characterized using atomic forcemicroscopy (AFM) and scanning electron microscopy (SEM). AFM imagingwill be performed using a Dimension Bioscope atomic force microscopeunder fluid conditions. Topographic images will be acquired in a tappingmode using silicon tips on integral cantilevers with a nominal springconstant of 20-100 N/m. Images will be obtained from at least twodifferent samples prepared on different days and at least threemacroscopically separate areas on each sample. Scan areas will rangefrom 10 nm×10 nm to 10 mm×10 mm. SEM images will be acquired with a Jeol6300FV equipped with a field emission gun (FEG). Because of the FEG,outstanding resolution can be achieved at accelerating voltages as lowas 0.5 keV which allows for imaging non-conducting materials such ascells without the need for coating. The point to point resolution at 1keV is 7 nm and decreases to 1.5 nm at 30 keV. The thickness andstoichiometry of the Ti-peptide hybrids will be determined withRutherford backscattering spectrometry. A 5SDH accelerator(electrostatics Corporation, Middleton Wis.) produces 0.5 to 5 MeV alphaparticles that impinge on the sample. The energies of the backscatteredparticles are detected by a solid-state detector with an energyresolution of 15 keV. The energy is converted to depth using RUMP(Computer Graphic Service, Ithaca N.Y.) software with a depth resolutionranging from 10 nm to 1 nm depending on the scattering geometry. Thethickness and stoichiometry of the titanium oxide as well as impurities(˜atomic percent) can be determined without standards. The concentrationand spatial distribution of N (amine), 0 (oxide) and C (APTS) will bedetermined by Auger Electron Spectroscopy (AES). AES spectra will beobtained with a Perkin-Elmer Phi 600 Scanning Auger multiprobe system.The sample is irradiated with 3 keV electron beam at current of 0.1 mA.Lateral resolution is 100 nm. Surface charge will be evaluated withcontact-angle goniometry. Static water contact angles will be measuredusing a home built contact angle goniometer. A 2 mL droplet of waterwill be suspended from the tip of a microliter syringe supported abovethe sample stage. The image of the droplet will be captured and thecontact angle measured using ATI Multimedia and Scion Image programs.

RGD (SEQ ID NO: 1) Quantification. Peptide distribution will bequantitated in two ways. The first will be to use radiolabeled RGD (SEQID NO: 1). Briefly, RGD (SEQ ID NO: 1)-containing peptides will beradiolabeled with 125I. Radiolabeled RGD (SEQ ID NO: 1)-containingpeptides will be incorporated into the surface as described above.Peptide distribution and density will be determined by autoradiographyand by scintillation counting. The second method will be to usefluorescently labeled RGD (SEQ ID NO: 1). Briefly, commerciallysynthesized peptides will be produced with a fluorescent tag in place ofthe terminal chloroacetyl group. The peptides purchased from Sigma willbe fluorescently labeled using the Alexa Fluor labeling kit (MolecularProbes, Eugene, Oreg.). Peptides will be column separated and thenresolved using ascending BETLC (72). Purified peptides will then beincorporated into the Ti discs as described above and quantitated interms of density and distribution using confocal microscopy.

Detection of amount and density of acid-labile tigecyclin on Ti may bedetermined by spectrofluorimetry and by epifluorescence microscopy.Tethered tigecyclin will be imaged using its own inherent fluorescence.Quantities will be estimated by comparison with known amounts oftigecyclin in the fmol-nmol range. Digital images will be collectedusing a Noran Instruments confocal scanning laser microscope which wehave successfully used in the past to image Ti surfaces. The confocalityallows filtering of the out-of-phase reflected light from the metalsurface.

The Ti-tigecyclin hybrids may be incubated in PBS (pH 5.0-8.0), weakacid, for 1-180 min. The PBS pH will be adjusted using the H₃PO₄:NaHPO₄ratio, while holding ionic strength constant. The results of this testwill indicate the most “active” pH's (for example pH 5.0, 5.5, 6.0 aswell as physiological pH (7.4)) for release of the tethered molecules.The release may be determined in a time course where antibiotic releaseis measured at 1, 2, 3, and 24 h.

Bactericidal activity of tethered tigecyclin against S. aureusinfections. The bactericidal activity of these surfaces will be testedagainst an initial inoculum of 10³-10⁶ S. aureus, a concentration thatwill occupy less than 1% of the 177 mm² modified Ti disk used for theseexperiments. Modified or unmodified discs will be sterilized by exposureto UV light and placed into the well of a 24-well tissue culture plate.0.5 mL of DMEM containing 10% FBS will be added and medium will beinoculated with 10³-10⁶ infectious units of S. aureus. The inoculum willbe mixed by agitation on a rotary shaker for 5 minutes, followed byincubation at 37° C. under normal tissue culture conditions, i.e., in a5% CO₂, humidified incubator with no agitation. Bacteria proliferationwill be measured as a function of time (1-24 h). Control surfaces willtest the proliferation of the bacteria on the Ti alone, the effect ofsoluble tigecyclin on bacterial propagation and the effect of the linkeralone. The proliferation curve of the bacteria will be determined every60 minutes for 4 h by extraction of the DNA and measurement using PCR.Non-adherent bacteria will be collected, pelleted, and resuspended in100 mL PBS. Adherent bacteria on the surface will be bathed in 100 mLPBS. Cell lysis will be achieved by four freeze/thaw cycles, followed bycentrifugation. The supernatants will be used for determination ofbacterial number by PCR. Briefly, 10-20 mL of the above DNA solutionswill be used in a 100 mL reaction volume using a Tris-KCl—MgCl₂ bufferand AmpliTaq, as known to those skilled in the art. Primers are directedat the 16S rRNA (Forward: CGGCAGGCCTAACACATGCAAGTCG. Reverse:GGTTGCGGCCGTACTCCCCAGG) to yield an 881 bp product, after 30-35 cycles.Detection will be by ethidium bromide staining after fractionation byagarose gel electrophoresis. In addition, pH measurements will beperformed to determine if bulk changes in pH are occurring as a resultof bacterial propagation. In parallel, bacterial number will be measuredby direct counting after plating serial dilutions on agar plates.Supernatant will be removed from cultures containing S. aureus as aboveand the surfaces will be washed with 2 mL of DMEM and pooled with thesupernatant from the same culture. Following agitation, this supernatantwill be plated on LB agar plates at dilutions of 0, 102, 104, and 106for determination of bacterial numbers. Additional dilutions will beused, as appropriate, to yield a countable number of colonies on theplate. Molecular Probes “Live/Dead BacLight” kit can be used tovisualize bacterial viability based on differential staining. Using theLive/Dead BacLight system (Molecular Probes), live and dead bacteriawill be stained with SYTO9+propidium iodide. Live cells will fluorescegreen, whereas cells with compromised membranes will fluoresce red.Digital images will be recorded and ratios of live:dead bacteriadetermined in random fields using double immunofluorescence.

Biocompatibility of the tethered tigecyclin. Biocompatibility will bedetermined by culturing MC3T3 osteoblast-like cells on the tetheredantibiotic surface. MC3T3-E1 cells will be maintained in DMEM containing10% FBS, 25 mg/mL ascorbate, and 5 mM β-glycerophosphate (formineralization studies). At 1, 5, and 10 days, numbers of adherent cellswill be determined by the BCECF-AM assay and actual cell numbers will bemeasured by DNA analysis. To assess development of the osteoblasticphenotype the expression of Osf-2, osteocalcin, and collagen type I willbe evaluated by RT-PCR. The functional status of the cells at 7 and 14days will be determined by measuring the activity of alkalinephosphatase. The mineralization potential will be evaluated by examiningthe cell layer for evidence of apatite formation by FT-IR.

Bactericidal activity and biocompatibility of tethered tigecyclin inpresence of cells. The modified surface should be permissive toosteoblastic cell proliferation and only release antibiotic in thepresence of bacteria. Based on the results of the studies describedabove, two concentrations of bacteria will be co-cultured with MC3T3-E1osteoblast-like cells. Effects on osteoblast phenotype and function willbe assessed as described, and bacterial survival will also be evaluated.

The outcome measures include the surface density of the modifiedtigecyclin tethered to Ti and the composition of the tigecyclinderivative that is attached to the surface. The overall efficiency ofattachment of tigecyclin to the Ti surface may be 80-90%. As only oneamide group has sufficient reactivity to be modified in the proposedreaction, it is expected that predominantly one reaction product, namelythe one described herein will be formed. This prediction will be testedby MALDI-TOF-mass spectrometry. If other groups are derivatized thenalternative protecting groups will be used to ensure that only thatamide is available for reaction with the Ti-linker-terminal amine. Afterattachment of tigecyclin to the Ti surface, extensive washing procedureswill be employed, making it unlikely that the surface will containactive, unmodified, untethered antibiotic. In the case of less thanstoichiometric coupling, some linker alone will be found tethered ontothe metal surface. Since it is expected that the treatments will resultin 80-90% modification, the concentration of the unreacted linker willbe small compared to the concentration of the modified tigecyclin.Should the actual density of the antibiotic on the Ti surface benon-uniform, then additional passivation techniques will be employed toensure a uniform, fresh Ti surface. For the acid-labile linkage, it isexpected that acidic solutions will cause significant and time-dependentrelease of tigecyclin. In the neutral pH range, there should be minimalrelease of the antibiotic. Should either acid labile linkage release tooquickly, the linkage may be modified to decrease its electron density,making it less likely to be protonated. The linkages may be used totether other antibiotics to the Ti surface, as new antibiotics aregenerated. These procedures result in a delivery system for theincorporation of bioactive substances into solid synthetic surfaces.

Bacterial proliferation should be retarded by the RGD (SEQ ID NO:1)/antibiotic-modified surfaces. The determination of the concentrationsof antibiotic needed to produce a bactericidal effect in an essentiallytwo-dimensional system will be determined from the experiment describedvide supra. Based on the antibiotic occupying ˜40 nm² of space, if anadherent bacterium occupies 1 mm², it will have ˜25,000 molecules ofantibiotic tethered for release/interaction directly at its surface.Based on preliminary data these numbers of tethered antibiotics shouldbe sufficient to eradicate a bactericidal infection. The experimentsdetailed allow measurement of the numbers of adherent and planktonicbacteria via PCR and colony formation assays, and also permit visualizethe live to dead ratio on the surface and in solution using dyesspecific for living or dead bacteria. If the number of tetheredantibiotic molecules is inadequate, multivalent linkages via the RGD(SEQ ID NO: 1)/APTS bonds may be used to increase tigecyclinconcentration.

The RGD (SEQ ID NO: 1) surface should enhance osteoblast maturation onthe Ti surface. The addition of antibiotics to the peptide-graftedsurface should not inhibit cellular adhesion or osteoblast maturation.This prediction is based on the fact that other antibiotic-containingsurfaces that have much higher concentrations of antibiotics immobilizedon their surfaces support such maturation. If the modified surfaceretards osteogenesis, different surface concentrations of the antibioticwill be used to increase ontogenesis.

The crystal structure of the RGD (SEQ ID NO: 1) loop region indicatesthat the motif is on an extended β hairpin-like loop within thefibronectin molecule. Based on this loop region, use of longer RGD (SEQID NO: 1) peptides may allow modification of the three dimensionalconfiguration to further promotes cell adhesion and maturation. In thiscase, it may be able to determine the required length of the loop regionthat is optimum for osteoblast binding to the Ti.

EXAMPLE 5

In this example the relationship between bacterial proliferation andbulk pH changes is used to illustrate that secretions from bacteria maybe used as an indication of cellular activity and result in release oftherapeutic molecules from a chemically modified surface.

To measure pH changes associated and bacterial proliferation, serum-freeDMEM was inoculated with E. coli and the bacterial proliferation andsolution pH monitored over the course of 6 hours at 37° C. in abacterial shaker. During this time, the bacteria were still in the logphase of growth and the pH of the solution decreased over 4 pH units,bringing the DMEM from its normal, slightly basic pH to a pH below 5.0.These results were reproducible. The acid released is an example of oneof several environmental cues that can be exploited in designingimplants with modified surfaces that release therapeutic molecules inresponse to the secretions of physiological state.

EXAMPLE 6

In this prophetic example method and composition are described that maybe used to couple a therapeutic molecule such as the antibiotic rifampinto an implant surface that includes a Ti-oxide surface. The coupling isfurther illustrated by using weak-acid-labile linkages. Method andprocedures used to characterize such modified implant surfaces and theirrelease kinetics are detailed.

Rifampin (C₄₃H₅₈N₄O₁₂, 822.95 Da) is an example of a large polycyclicantibiotic. The non-active 17-OH group of the antibiotic rifampin((2S*,12Z,14E,16R*,17R*,18S*,19S*,20S*,21R*,22S*,23R*,24E)-5,6,9,17,19,21-hexahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-8-[N-(4-methyl-1-piperazinyl)formimidoyl]-2,7-(epoxypentadeca[1,11,13]trienimino)naphtha[2,1-b]furan-1,11-deone21-acetate) may be modified by esterification with.2-methyl-4-thioethylamido maleic acid. This reaction may be monitored byTLC, using reactants as standards and the product characterized by HPLCand mass spectroscopy. The modified rifampin (sm-Rif) can be covalentlyattached to an activated, smooth Ti alloy surface via production of aTi—S or through use of APTS chemistry to produce a Ti—O—SiR bondedgroup. After reaction, the antibiotic surface density on the Ti as wellas the extent of immobilization may be characterized. The surfacedensity may be measured using a top-down microtiter plate reader withfluorescent capabilities (amounts can be determined by comparison withknown amount of rifampin dried onto a Ti surface). The distribution ofthe sm-Rif may be determined using eipfluorescence microscopy.Characterization of the moieties tethered to the surface can bedetermined by Matrix Assisted Laser Desoprtion Ionization-Time Of Flight(MALDI-TOF) mass spectroscopy. Characterization of the molecular ionsresulting from the fragmentation of the sm-Rif can be compared withvalues obtained from unmodified rifampin and published literaturevalues.

The surface linkage stability and release of a tethered molecule suchsm-Rif from the Ti may be determined as a function of time and pH.Measurement of the effect of pH on the release rates of the antibioticfrom the surface at different time periods can be made. The totalsolubilized rifampin released by the change in pH may be determined byspectrophotometry or spectrofluorimetry. Biological activity of thetethered molecules may be measured using a disc diffusion assay using alawn of S. aureus. In this assay, active antibiotic on a surfacederivatized or chemically modified implant produces a zone of growthinhibition around the disk and by comparison with known concentrationsof antibiotic, produces an estimate of the active concentration ofantibiotic on the surface of the implant.

EXAMPLE 7

In this prophetic example, specific conditions for the modification ofrifampin and attachment of the modified rifampin to a titanium disc aredisclosed.

All synthetic reactions can be performed with sterile solutions tominimize subsequent UV sterilization prior to cell culture. Rifampin canbe obtained from Sigma or from BIOMOL Research Laboratories, with anestimated purity of >95%.

Synthesis of smRif. 2-Thioethylamine can be reacted with 1.5 equivalentsof 2-methyl maleic anhydride in dry CH₃CN with 0.1 equivalents ofdimethylaminopyridine (DMAP) in a glove box with stirring under argon at25° C. until complete conversion to 2-methyl maleyl thioethylamide.Conversion can be measured by TLC on silica developed with 70% CHCl₃/30%MeOH. Unreacted 2-methyl maleic anhydride can be hydrolyzed by additionof aqueous NaHCO₃. The 2-methyl maleyl thioethylamide product can thenbe extracted twice from the solution with one volume of CHCl₃. Theorganic layer can be washed again with aqueous NaHCO₃, then added to twovolumes of pentane to precipitate the 2-methyl maleyl thioethylamideproduct. The product can be characterized by TLC and mass spectroscopy.Product that is at least 90% pure can be esterified in dry CHCl₃ to the17-OH of 0.5 equivalents of rifampin upon the addition of 1.2equivalents of N,N′-dicyclohexylcarbodiimide (DCC) with stirring underargon at 25° C. until complete conversion to17-(2-methyl-4-thioethylamido maleyl)rifampin (sm-Rif), assayed by TLCon silica developed with 50% CHCl₃/50% MeOH. The resultingN,N′-dicyclohexylurea can be sedimented, and the solution can then beextracted with one volume of diethyl ether. The CHCl₃ phase can then beadded to two volumes of pentane to precipitate the sm-Rif product. Theproduct can be characterized by C₁₈ reversed-phase HPLC and MALDI-TOFmass spectroscopy.

HPLC monitoring of product formation. Sm-Rif can be purified fromcontaminating rifampin and 2-methyl maleyl thioethylamide byargon-flushed reversed-phase liquid chromatography on a 10×250 mmAlltima C₁₈ column eluted with a gradient over 30 min. from 10%CH₃CN/90% H₂O to 90% CH₃CN/10% H₂O at 2 mL/min, monitored at 238 nm.

A fresh Ti surface can be produced by incubation of smooth Ti disks innitric acid to remove the oxide coating, followed by extensive rinsingwith de-gassed, oxygen-free PBS in a nitrogen atmosphere. The sm-Rif,dissolved in oxygen-free PBS can be reacted with the fresh Ti surface inan inert atmosphere for 30 min. at 25° C. The thiol bond to the Ti is afavorable reaction that proceeds with high efficiency in the absence ofoxygen. As a control, the 2-methyl maleyl thioethylamide precursor canalso be reacted with a fresh Ti surface.

Alternatively the sm-Rif, may be coupled to a titanium surface silylatedwith (3-glycidoxypropyl)trimethoxysilane (GPTS) to form a thioetherTi—O—Si(CH₂OH)(CH₂)—Sm-RIF

Non-hydrolyzable Ti-Rif can be synthesized by a similar esterificationroute, except that 3-thiopropanoic acid can be condensed with the 17-OHof rifampin followed by purification and characterization as above.Bonding to the Ti surface can be as above.

Detection of amount and density of rifampin on Ti by spectrophotometryor fluorimetry. Rifampin can be detected by fluorimetry after oxidation.Either the surface containing the immobilized rifampin or the solutioncontaining rifampin can be oxidized in 6% H₂O₂ in PBS for 5 min. Anequal volume of PBS, pH 7.9 can then be added and incubation cancontinue for 25 min., at which time rifampin fluorescence is optimal.Fluorescence persists for approximately an additional 30 min, limitingthe numbers of oxidations that can be performed at any one time.Oxidation and fluorimetric detection of rifampin on the surface can beused for determination of the amount of rifampin covalently bonded tothe implant surface. Measurement of solubilized rifampin can either usefluorimetry as above, with sensitivity in the 0.1 μg/mL range, orspectrophotometry (λ=334 nm, ε=27000 g-mole/1).

MALDI-TOF Mass Spectroscopy. Derivatized Ti disks overlaid with asinapinic acid matrix can be allowed to outgas under high vacuum in theCiphergen sample chamber for 30 min. before analysis. Molecular ionformation can be achieved through firing of a 338 nm laser onto thesurface of the Ti, causing bond breakage and release of the parent ion.Because of the innate charge of the molecular ions, the parent ion andany fragments can be accelerated into the mass spectrometer where thetime of flight can be a function of the mass of the molecular ions.Computer assignment of the identities of molecular ions by Ti-smRif canbe based upon reported values for rifampin and comparison with thepattern produced by underivatized rifampin alone.

The Ti-smRif hybrids can be incubated in PBS (pH 5.0-8.0) for 1-180 min.The PBS pH can be adjusted using the H₃PO₄: NaHPO₄ ratio, while holdingionic strength constant. The most “active” pH values, which are expectedto be pH 5.0, 5.5 and 6.0 for the present linkage, but which may bedifferent for other linkages, may be determined in a time course whereantibiotic release can be measured at 1, 2, 3 and 24 h. In parallel, thesurface can also be incubated in PBS, pH 7.4 to determine the releaserate under normal physiological pH.

Disc Diffusion Assay. Six mm sterile filter disks can be directly placedonto a full-grown lawn of S. aureus (ATCC 25923) culture, and knownvolumes (20 μL) and dilutions of the eluted antibiotic can be pipettedonto the disc. After incubation overnight, the area of the resultantadjacent clear zone that is devoid of bacterial growth can be measuredand compared with the area cleared by a standard series of known dosesof rifampin. A 1 μg/mL solution of rifampin should give a clear zone of4-5 cm. The lower limit of sensitivity of this assay is expected to bein the 10 ng/mL range.

After attachment of the antibiotics to the Ti surface, extensive washingprocedures can be employed, making it unlikely that the surface cancontain active, unmodified, untethered antibiotic. Measurement of activeantibiotic released under mildly acidic conditions from modified implantsubstrates may be measured with a disk diffusion assay using S. Aureusas the target bacteria.

EXAMPLE 8

In this prophetic example, procedures, methods, and compositions used todetermine if antibiotic release from compositions of the presentinvention lowers bacterial counts while sustaining osteoblastic cellproliferation and maturation are described.

In the establishment of an infection, the number of bacteria present atthe site is initially very low. The size of the initial inoculum inthese experiments can be determined by two parameters. Sufficientbacteria to be able to estimate bacterial number over several hoursusing PCR should be present. Using the PCR conditions known to thoseskilled in the art, it is possible to measure bacterial numbers greaterthan 1×10³. If the numbers of bacteria present are only sufficient tocoat a small area of the surface, it may be possible to assess theeffects of the surface modifications on a pre-monolayer. Assuming a sizeof 1-5 μm² for bacteria, 10³-10⁶ bacteria would occupy less than 1% ofthe total area of the disk for a 24-well plate and therefore should bewithin the test target. Inoculation will be made using about 10³-10⁶ S.aureus into 0.5 mL DMEM containing 10% FBS.

Using this inoculum of cells, the bactericidal activity of the Ti-smRifsurface or other functionalized surfaces can be tested. Three controlsurfaces, unmodified Ti±1 μg/mL Rif (in the bathing medium), Ti-modifiedwith the linker alone (Ti-sm), and Ti-modified with irreversibly-bondedrifampin (Ti-Rif) can be tested in parallel with the Ti-smRif using DMEMcontaining 10% FBS as the bathing medium. The control surfaces can testthe proliferation of the bacteria on the Ti alone, the effect of solubleRif on bacterial propagation, the effect of the linker alone (which isthe moiety that can be left after bond cleavage) on osteoblast andbacterial proliferation, and finally the effect of irreversiblyimmobilized rifampin on bacterial proliferation, respectively. Theproliferation curve of the bacteria over the course of about 4 hours canbe determined every 60 minutes by extraction of the DNA and measurementusing PCR. Numbers of bacteria can be estimated by comparison with PCRsignals using DNA isolated from known numbers of bacteria. In addition,pH measurements can be performed as a result of bacterial propagation.Bacterial number can also be measured by direct counting. Serialdilutions of the bacteria can be made and plated onto LB agar. Colonyformation can be assessed by cell counting after photography and totalnumbers of colonies calculated. These values can be compared with thenumbers determined by PCR. This comparison can ensure that the numbersdetermined in the PCR experiments reflect bacteria capable of furtherproliferation (or under optimal conditions, represent a true cure of thebacterial infection).

Using two concentrations of bacteria that will be killed when incubatedwith the active surface, the effects of co-culture with Saos-9osteoblast-like cells can be determined. These cells can be plated at adensity of 1×10⁴ cells/1.77 cm² disk at the same time as inoculation ofbacteria. It can then determine if the Ti-smRif or other bondedantibiotic surface impedes the bacteria's adhesion, proliferation, andmaturation. Measurement of osteoblast number and expression of theosteoblastic cell phenotype may be continued for about 14 days so thatfunctional indicators, including the activity of alkaline phosphataseand mineral formation can be evaluated. Control conditions can includeosteoblastic cells on the Ti and Ti-sm surfaces.

EXAMPLE 9

In this prophetic example specific procedures and conditions that may beused to determine if antibiotic release from modified implant surface ofthe present invention lowers bacterial counts while sustainingosteoblastic cell proliferation and maturation.

Modified or unmodified discs can be sterilized by exposure to UV lightand placed into the well of a 24-well tissue culture plate. 0.5 mL ofDMEM containing 10% FBS can be added and medium can be inoculated withprobably 10³-10⁶ infectious units of S. aureus. The inoculum can bemixed by agitation on a rotary shaker for 5 minutes, followed byincubation at 37° C. under normal tissue culture conditions, i.e. in a5% CO₂, humidified incubator with no agitation. Bacteria proliferationcan be measured as a function of time (1-24 h).

Non-adherent bacteria can be gathered in the supernatant, pelleted, andresuspended in 100 μL PBS. Adherent bacteria on the surface can bebathed in 100 μL PBS. Cell lysis can be achieved by four freeze/thawcycles. Freezing can be in dry/ice ethanol and thawing at 65° C., for 1min for each cycle. DNA from the adherent samples can be transferred toan Eppendorf tube, and the disk further rinsed with an additional 100 μLof PBS to remove any DNA still adherent to the surface. After thefreeze/thawing cycles, the samples can be centrifuged in a microfuge fortwo minutes and the supernatants can be used for determination ofbacterial number by PCR.

PCR may be used to determine bacterial number. Briefly, 10-20 μL of theabove DNA solutions can be used in a 100 μL reaction volume using aTris-KCl—MgCl₂ buffer and AmpliTaq. Primers are directed at the 16S rRNA(Forward: CGGCAGGCCTAACACATGCAAGTCG. Reverse: GGTTGCGGCCGTACTCCCCAGG) toyield an 881 bp product. PCR conditions are as follows: denaturation:94° C., 1 min. Annealing: 55° C., 1 min. Extension: 72° C., 2 min for30-35 cycles. Detection can be by ethidium bromide staining afterfractionation by agarose gel electrophoresis.

Determination of S. aureus numbers by dilution. Supernatant can beremoved from cultures containing S. aureus and the surfaces can bewashed with 2 mL of DMEM and pooled with the supernatant from the sameculture. Following agitation, this supernatant can be plated on LB agarplates at dilutions of 0, 10², 10⁴, and 10⁶ for determination ofbacterial numbers. Additional dilutions can be used, as appropriate, toyield a countable number of colonies on the LB surface. Surfaces can bephotographed and cell numbers determined by direct cell counting.Determinations can be performed by two independent investigators.

Determination of Saos-9 cell adhesion, proliferation, and maturation.The Ti can be harvested, washed three times in PBS to remove bacteria.The cells can be released from the Ti surface by treatment with 0.05%trypsin for 5 min, and collected by centrifugation at 200×g for 10 min.Cells can be washed and resedimented three times. This procedure willremove almost all of the adherent bacteria. Numbers of adherent cellscan be determined by the BCECF-AM assay. Numbers of the viable Saos-9cells can be determined by MTT assay; actual cell numbers can bemeasured by DNA analysis. To assess development of the osteoblasticphenotype, we can evaluate the expression of Osf-2, osteocalcin, andcollagen type I by RT-PCR. The functional status of the cells at 5, 10,and 14 days can be determined by measuring the activity of alkalinephosphatase. The mineralization potential can be evaluated by including5 mM β-glycerophosphate in the culture medium and by examining the celllayer for evidence for apatite formation by FTIR.

EXAMPLE 10

This example illustrates the immobilization of bioactive peptides tometal surfaces that can be bonded to other therapeutic molecules andthat can result in osteoblast cell maturation due to the RGD (SEQ IDNO: 1) amino acid sequence.

The peptide RGD (SEQ ID NO: 1) was couple to surfaces using the silanesurface chemistry. The impact of the peptide coupling on the surface wasexamined using atomic force microscopy and by the effect of the peptideon the differentiation of attached osteoblasts. The atomic forcemicrographs illustrate that the treatment causes a small change insurface roughness of the substrate. It was observed that cells wereimmediately bound to the membrane and proliferated. After 8 days on thematerial surface, osteoblasts exhibited high levels of alkalinephosphatase staining, indicating that the cells were undergoingmaturation. Alizarin red staining and Fourier transform infrared (FTIR)spectroscopy showed that the mineral formed by the cells was abiological apatite. Control cells, maintained on the tripeptide RGE, didnot exhibit these effects within the time period evaluated. This exampledemonstrates that even though the peptide tether contains only threeamino acids, it retains its bioactivity on the material surface.

EXAMPLE 11

This prophetic example demonstrates preparation and characterization ofa surface with therapeutic molecules covalently bonded to an implantsurface that enhances bone cell attachment and thereby facilitatestissue-implant integration, while remaining a long-term reservoir oftherapeutic oligonucleotides to eradicate any recurrence of osteosarcomaor other cancers that arise locally or from hematogenous sources, i.e.,metastatic cells.

For enhanced osseointegration, the RGD (SEQ ID NO: 1) peptide motif canbe used as in Example 4 above. The implant can include covalently linkedRGD (SEQ ID NO: 1)-containing peptides to a Ti surface, creating apeptide-Ti hybrid. This can provide a mixed surface as some of the RGD(SEQ ID NO: 1) motifs will serve as part of the linker for theimmobilization of the therapeutic oligonucleotides. The RGD (SEQ ID NO:1)-oligonucleotide linkage will be labile to matrix metalloproteinasesreleased by cancer cells, thereby exposing additional RGDs (SEQ IDNO: 1) for promotion of osseointegration as the therapeuticoligonucleotide is released. Peptide sequences specifically cleaved bymatrix metalloproteinases will be placed between the RGD (SEQ ID NO: 1)motif and the therapeutic oligonucleotide. The effect of such flankingamino acid sequences may be determined by progressively increasing theflanking amino acids at each side of the RGD (SEQ ID NO: 1) motif andanchor on the modified peptides covalently bonded to the Ti surface.

Therapeutic oligonucleotides targeted against the MYC oncogeneconstitute one example of a broad anticancer oligonucleotide. Thetherapeutic oligonucleotides may be composed of various combinations ofmodified bases, modified sugars, and modified internucleotide linkages.The therapeutic oligonucleotide targeted against the MYC oncogene mayinclude peptide nucleic acid (PNA) residues linked toTi—O—Si(CH₂)₃NH-RGD (SEQ ID NO: 1) by a peptide sequence specificallycleaved by matrix metalloproteinases, and a basic peptide sequenceconducive to cellular uptake. The metalloproteinase-susceptible linkerwill be cleaved by matrix metalloproteinases secreted by cancer cellsgrowing at the site of the implant. The liberated basic peptide-PNAchimera will be taken up quickly into the cytoplasm of the cancer cells,where it will inhibit the translation of MYC mRNA. As a result,production of Myc protein will be significantly reduced. Because Mycprotein plays a vital role in cell proliferation, growth of theimmediately neighboring cancer cells will be reduced.

Ti alloy (Ti6Al4V) surfaces for the implant will be obtained, modified,and characterized as described in Example 4.

The modified Ti surfaces can be evaluated for oligonucleotide releasekinetics in the presence of osteosarcoma cells in culture, and forantiproliferative activity against the osteosarcoma cells. The modifiedsurface may also be characterized to determine if the modified surfacesupports osteoblast proliferation and maturation both in the presenceand absence of metastatic cancer cells.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. For example, in the immediate future, we can concentrateon defining the conditions that increase the stability of the surfacewhile maintaining sensitivity to environmental cues such or cellsecretions. The acid-labile linkage is only one of example of a linkageuseful in the present invention. For instance, the biofilm slime iscomprised of exopolysaccharides that could also be used to destabilize atethering linker, as could enzymes secreted from the bacteria to modifytheir environment for biofilm formation. Therefore the spirit and scopeof the appended claims should not be limited to the description and thepreferred versions contain within this specification.

1-38. (canceled)
 39. An implantable article comprising: an implant for amammal having a biologically compatible surface, wherein at least aportion of said surface is silylated with organosilanes; a linker havinglinking groups wherein a terminus of a linking group is covalentlybonded to one or more of said organosilanes and wherein said linker isat least one of oligo(ethylene glycol) and an acid labile bond; and atleast one therapeutic molecule, wherein said therapeutic molecule isbonded to said linking group or linking groups and wherein saidtherapeutic molecule interacts with cells adjacent to the surface ofsaid implant.
 40. The implantable article of claim 39 wherein saidtherapeutic molecule enters a membrane of said cells or passes through awall of said cells.
 41. The implantable article of claim 39 wherein thetherapeutic molecule is a member selected from the group consisting ofpeptide, therapeutic oligonucleotides, an antibiotic, cell growthfactors, chemotherapeutics, thrombolytic, anti-inflammatories, andosteoactive factors.
 42. The implantable article of claim 39 whereinsaid therapeutic molecule includes a peptide.
 43. The implantablearticle of claim 42 wherein labilization of the therapeutic moleculefrom the linker leaves a peptide covalently bonded to the surface thatpromotes the adhesion and maturation of cells.
 44. The implantablearticle of claim 39, where the linker includes oligo(ethylene glycol).45. The implantable article of claim 39 wherein the implant is made fromat least one of titanium, titanium alloy, tantalum, CoCrMo alloys,stainless steels, cobalt, chromium, aluminum, zirconium gold, siliconand their alloys, TEFLON/PTFE, polyethylene, ultra high molecular weightpolyethylene, silicone, silica, glass, polystyrene and zirconia.
 46. Theimplantable article of claim 39 wherein said linker includes the acidlabile bond, and said therapeutic molecule is released from said linkerby interacting with said cells.
 47. The implantable article of claim 39wherein the therapeutic molecule interacts with said cells to alterangiogenesis, or decrease bacterial proliferation adjacent to saidimplant.
 48. The implantable article of claim 39 wherein saidtherapeutic molecule is cleaved from said linker by intracellularenzymes or acid.
 49. The implantable article of claim 39 wherein saidtherapeutic molecule is exchanged with endogenous ligands of the cell.50. The implantable article of claim 39 wherein said acid labile linkeris a member selected from the group consisting of methylmaleamide,hydrazone, and combinations thereof.
 51. The implantable article ofclaim 41 wherein labilization of the antibiotic from the linker providesthe peptide that promotes the adhesion and maturation of bone cells. 52.The implantable article of claim 41 wherein the antibiotic is a memberselected from the group consisting of minocyclins, tigecyclin,glycylcycline, vancomycin and its analogs, rifampin and its analogs,gentamycin and its analogs, or combinations thereof.
 53. The implantablearticle of claim 41 wherein said implant promotes osseointegration. 54.The implantable article of claim 41 wherein said therapeutic molecule isa peptide and an antibiotic and wherein the antibiotic is competitivelybonded to said linker.
 55. The implantable article of claim 46 whereinsaid therapeutic molecule includes a peptide.
 56. A method of treating amammal comprising: inserting an implant into a site in need thereof onsaid mammal, said implant having a biologically compatible surfacewherein at least a portion of said surface is silylated withorganosilanes; a linker having linking groups wherein a terminus of alinking group is covalently bonded to one or more of said organosilanesand wherein said linker is at least one of oligo(ethylene glycol) and anacid labile bond; and at least one therapeutic molecule, wherein saidtherapeutic molecule is bonded to said linking group or linking groupsand wherein said therapeutic molecule interacts with cells adjacent tothe surface of said implant.
 57. The method of claim 56 wherein saidtherapeutic molecule is a member selected chosen from the groupconsisting of peptide, therapeutic oligonucleotides, antibiotics, cellgrowth factors, chemotherapeutics, thrombolytic, anti-inflammatories,and osteoactive factors.
 58. The method of claim 56 wherein said implantis used for fracture fixation.
 59. The method of claim 56 wherein theimplant promotes osseointegration.
 60. The method of claim 56 whereinthe therapeutic molecule prevents bacterial proliferation.
 61. Themethod of claim 56 wherein said therapeutic molecule is released fromsaid linker by reaction with a cellularly derived acid, an enzyme, orligand.
 62. The method of claim 56 wherein said implant alters theproliferation of cells at the site of said implant.
 63. The method ofclaim 56 wherein said implant is at least one of intramedullary nail anda prosthesis.